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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230597	1/9	Homo sapiens	ESC	(d)(U)C DG	Chen Z	2024	100%
Study summary Full title Identification of specific markers for human pluripotent stem cell-derived small extracellular vesicles. All authors Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y Journal J Extracell Vesicles Abstract Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Adj. k-factor 37432 (washing) Protein markers EV: Alix/ CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics yes EV density (g/ml) 1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ESC EV-harvesting Medium Serum free medium Cell count 2.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 25 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 37432 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 20% Highest density fraction 60% Total gradient volume, incl. sample (mL) 11 Sample volume (mL) 1 Orientation Top-down Speed (g) 150000 Duration (min) 600 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 10018 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101 Not detected contaminants GM130/ Calnexin Flow cytometry Type of Flow cytometry Nano-flow Cytometry Hardware adaptation to ~100nm EV's By utilizing Rayleigh scattering Calibration bead size 0.25 Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Nano-flow Cytometry Hardware adjustment By utilizing Rayleigh scattering Calibration bead size 68/ 91,113,155 Report type Size range/distribution Reported size (nm) 40-150 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.03E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV230597	2/9	Homo sapiens	iPSC	(d)(U)C DG	Chen Z	2024	100%
Study summary Full title Identification of specific markers for human pluripotent stem cell-derived small extracellular vesicles. All authors Chen Z, Luo L, Ye T, Zhou J, Niu X, Yuan J, Yuan T, Fu D, Li H, Li Q, Wang Y Journal J Extracell Vesicles Abstract Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinic (show more...)Pluripotent stem cell-derived small extracellular vesicles (PSC-sEVs) have demonstrated great clinical translational potential in multiple aging-related degenerative diseases. Characterizing the PSC-sEVs is crucial for their clinical applications. However, the specific marker pattern of PSC-sEVs remains unknown. Here, the sEVs derived from two typical types of PSCs including induced pluripotent stem cells (iPSC-sEVs) and embryonic stem cells (ESC-sEVs) were analysed using proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and surface marker phenotyping analysis by nanoparticle flow cytometry (NanoFCM). A group of pluripotency-related proteins were found to be enriched in PSC-sEVs by LC-MS/MS and then validated by Western Blot analysis. To investigate whether these proteins were specifically expressed in PSC-sEVs, sEVs derived from seven types of non-PSCs (non-PSC-sEVs) were adopted for analysis. The results showed that PODXL, OCT4, Dnmt3a, and LIN28A were specifically enriched in PSC-sEVs but not in non-PSC-sEVs. Then, commonly used surface antigens for PSC identification (SSEA4, Tra-1-60 and Tra-1-81) and PODXL were gauged at single-particle resolution by NanoFCM for surface marker identification. The results showed that the positive rates of PODXL (>50%) and SSEA4 (>70%) in PSC-sEVs were much higher than those in non-PSC-sEVs (<10%). These results were further verified with samples purified by density gradient ultracentrifugation. Taken together, this study for the first time identified a cohort of specific markers for PSC-sEVs, among which PODXL, OCT4, Dnmt3a and LIN28A can be detected with Western Blot analysis, and PODXL and SSEA4 can be detected with NanoFCM analysis. The application of these specific markers for PSC-sEVs identification may advance the clinical translation of PSCs-sEVs. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Adj. k-factor 37432 (washing) Protein markers EV: TSG101/ CD9/ CD63/ CD81 non-EV: None Proteomics yes EV density (g/ml) 1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells iPSC EV-harvesting Medium Serum free medium Cell count 2.00E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 25 Wash: time (min) 70 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 37432 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 20% Highest density fraction 60% Total gradient volume, incl. sample (mL) 11 Sample volume (mL) 1 Orientation Top-down Speed (g) 150000 Duration (min) 600 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 10 Pelleting: speed (g) 150000 Pelleting: adjusted k-factor 10018 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD9/ CD63/ TSG101 Not detected contaminants GM130/ Calnexin Flow cytometry Type of Flow cytometry Nano-flow Cytometry Hardware adaptation to ~100nm EV's By utilizing Rayleigh scattering Calibration bead size 0.25 Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database ProteomeXchange Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type Nano-flow Cytometry Hardware adjustment By utilizing Rayleigh scattering Calibration bead size 68/ 91,113,155 Report type Size range/distribution Reported size (nm) 40-150 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.03E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 100

	EV230372	7/14	Homo sapiens	H2228	(d)(U)C DG	Schöne N	2024	100%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large EVs Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H2228 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ Mitofilin/ Arf6/ CD81 Not detected EV-associated proteins Syntenin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 196.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.83E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	9/14	Homo sapiens	H596	(d)(U)C DG	Schöne N	2024	100%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large EVs Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ Mitofilin/ Arf6 Not detected EV-associated proteins Syntenin/ CD81 Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 188.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.33E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	13/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	100%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NSCLC Focus vesicles large EVs Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Actinin-4/ Rgap1/ EMMPRIN/ EpCAM/ EGFR non-EV: ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ EMMPRIN Detected contaminants ApoB Not detected contaminants ApoA1 Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 187 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.05E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220300	3/18	Homo sapiens	MCF7	UF (d)(U)C Filtration DG SEC (non-commercial)	Pinheiro C	2024	100%
Study summary Full title Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data. All authors Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, Van Dorpe S, De Craene B, Berx G, Boterberg T, Sys G, Denys H, Miinalainen I, Mestdagh P, Vandesompele J, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diver (show more...)Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration (Differential) (ultra)centrifugation Filtration Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ CD9/ Flotillin-1/ TSG101/ Syntenin non-EV: Argonaute 2 Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Validation of standards Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 96 Cell count 4.43e8 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 15.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 _m Ultra filtration Cut-off size (kDa) 10 kDa Membrane type Regenerated cellulose Size-exclusion chromatography Resin type EV-subtype Distinction between multiple subtypes Size Used subtypes 120-250 Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) particles per milliliter of starting sample: 1.54E11-2.60E11 Western Blot Detected EV-associated proteins Alix/ CD9/ Flotillinð1/ TSG101/ Syntenin Not detected contaminants Argonauteð2 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 2 RNAse treatment Yes RNAse type RNase A RNAse concentration 8 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125.7-134.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.54E11-2.60E11 EM EM-type Transmission-EM Image type Wide-field

	EV220300	4/18	Homo sapiens	A549	UF (d)(U)C Filtration DG	Pinheiro C	2024	89%
Study summary Full title Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data. All authors Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, Van Dorpe S, De Craene B, Berx G, Boterberg T, Sys G, Denys H, Miinalainen I, Mestdagh P, Vandesompele J, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diver (show more...)Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications. (hide) EV-METRIC 89% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: Alix/ CD9/ TSG101 non-EV: None Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Validation of standards Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A549 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 93 Cell count 1.09e8 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW32.1 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 15.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 _m Ultra filtration Cut-off size (kDa) 10 kDa Membrane type Regenerated cellulose Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) particles per milliliter of starting sample: 9.88E10 Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 186.1 EV concentration Yes Particle yield particles per milliliter of starting sample: 9.88E10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230598	1/3	Homo sapiens	Milk	(d)(U)C	Ten-Doménech, Isabel	2024	78%
Study summary Full title Normalization approaches for extracellular vesicle-derived lipidomic fingerprints – A human milk case study All authors Isabel Ten-Doménech, Victoria Ramos-Garcia, Abel Albiach-Delgado, Jose Luis Moreno-Casillas, Alba Moreno-Giménez, María Gormaz, Marta Gómez-Ferrer, Pilar Sepúlveda, Máximo Vento, Guillermo Quintás, Julia Kuligowski Journal Chemometrics and Intelligent Laboratory Systems Abstract Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a li (show more...)Human milk (HM) extracellular vesicles (EVs) are nano-sized, cell-derived particles sheathed in a lipid bilayer that encase specific cargo for delivery from mother to infant. The aim of this study was to expand our understanding of the lipidomic fingerprint of HM-EVs, with a specific focus on the impact of data normalization using simulated and experimental data obtained from the analysis of HM samples from mothers of preterm (N = 5) and term infants (N = 5), and a pool of donor human milk from 20 mothers (before and after pasteurization). EVs were isolated by multi-stage ultracentrifugation and characterized in terms of total protein content, total particle count and size, surface tetraspanin profile and protein markers, and morphology. Lipidomic analysis after single-phase extraction was performed by liquid chromatography mass spectrometry (LC-MS). The effect of widely used data normalization strategies (i.e., sample volume, particle count, protein content, total lipids signal) was compared. Results show that for the selection of the optimum normalization approach, the specific study aims, as well as the purity and homogeneity of size distribution of EV isolates should be considered. While normalization attending the particle number can be useful for between sample comparisons in EV populations with similar particle size, normalization to total lipid content is preferred when lipid contamination is encountered. Our findings exemplify the need for guidance with respect to data processing in LC-MS-based lipidomics studies of EVs. (hide) EV-METRIC 78% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin Mothers of preterm infants Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ HSP70 non-EV: Calnexin Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 50.2 Ti Pelleting: speed (g) 108763 Wash: volume per pellet (ml) 25 Wash: time (min) 120 Wash: Rotor Type Type 50.2 Ti Wash: speed (g) 108763 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of EV isolate Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168-238 EV concentration Yes Particle yield as number of particles per milliliter of EV isolate: 1.4E10-1.4E12 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(1) ExoView Report type Mean Report size 58-70 EV-concentration Yes Particle yield as number of particles per milliliter of EV isolate: 6E07-3E08

	EV230372	3/14	Homo sapiens	HCC-78	(d)(U)C DG	Schöne N	2024	78%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large EVs Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCC-78 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 17000 Wash: volume per pellet (ml) 1 Wash: time (min) 30 Wash: Rotor Type Heraeus 3331 Wash: speed (g) 17000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Actinin-4/ Rgap1/ Syntenin-1/ Arf6/ Mitofilin Not detected EV-associated proteins CD81 Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM/ EGFR Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 200.13 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.63E+09

	EV230372	8/14	Homo sapiens	H2228	(d)(U)C	Schöne N	2024	78%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small EVs Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H2228 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Syntenin/ Arf6/ CD81 Not detected EV-associated proteins Actinin-4/ Rgap1/ Mitofilin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 163.8 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.10E+10 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230372	10/14	Homo sapiens	H596	(d)(U)C DG	Schöne N	2024	78%
Study summary Full title PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer. All authors Schöne N, Kemper M, Menck K, Evers G, Krekeler C, Schulze AB, Lenz G, Wardelmann E, Binder C, Bleckmann A Journal J Extracell Vesicles Abstract Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). (show more...)Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45 /CD62P lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1 lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small EVs Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD81/ Syntenin/ Actinin-4/ Arf6/ Rgap1/ Mitofilin non-EV: GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Proteomics no EV density (g/ml) 1.10-1.17 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H596 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 143000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 90 Wash: Rotor Type TLA-55 Wash: speed (g) 143000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Pelleting-wash: volume per pellet (mL) 16 Pelleting-wash: duration (min) 60 Pelleting-wash: speed (g) SW 32 Ti Characterization: Protein analysis Protein Concentration Method Lowry-based assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Syntenin/ Actinin-4/ Arf6/ CD81 Not detected EV-associated proteins Rgap1/ Mitofilin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 141.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.09E+09 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220326	1/4	Homo sapiens	Glioblastoma stem-like cells NCH421k	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH421k EV-harvesting Medium Serum free medium Cell viability (%) 91 - 93 Cell count 194200000 - 222312000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 (RPLP0) Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175.0 EV concentration Yes Particle yield particles per milliliter of starting sample: 4,58E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220326	2/4	Homo sapiens	Glioblastoma stem-like cells NCH644	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH644 EV-harvesting Medium Serum free medium Cell viability (%) 83 - 90 Cell count 225576000 - 231070000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 169.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 1,87E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220326	3/4	Homo sapiens	Glioblastoma stem-like cells NCH705	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH705 EV-harvesting Medium Serum free medium Cell viability (%) 98 Cell count 204312000 - 225840000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 (RPLP0) Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 179.7 EV concentration Yes Particle yield particles per milliliter of starting sample: 6,05E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220326	4/4	Homo sapiens	Glioblastoma stem-like cells NCH711d	(d)(U)C DC	Lokumcu T	2024	78%
Study summary Full title Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. All authors Lokumcu T, Iskar M, Schneider M, Helm D, Klinke G, Schlicker L, Bethke F, Müller G, Richter K, Poschet G, Phillips E, Goidts V Journal ACS Nano Abstract Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-l (show more...)Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles small extracellular vesicles (sEVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 non-EV: Albumin/ Calreticulin/ Calnexin/ Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Cytochrome c1/ Lamin B1/ 60S acidic ribosomal protein P0 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Glioblastoma stem-like cells NCH711d EV-harvesting Medium Serum free medium Cell viability (%) 89 - 91 Cell count 102240000 - 116112000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 100000 Density cushion Density medium Iodixanol Sample volume 7 Cushion volume 4 Density of the cushion 20% Centrifugation time 70 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per million cells Western Blot Detected EV-associated proteins Alix/ CD9/ TSG101/ Syndecan-1/ Enolase 1 Detected contaminants 60S acidic ribosomal protein P0 (RPLP0) Not detected contaminants GM130/ Lamin B1/ Cytochrome c Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ Calnexin Not detected contaminants Argonaute 2/ GM130/ PMP70/ Tamm-Horsfall protein/ Cytochrome c1 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 203.5 EV concentration Yes Particle yield particles per milliliter of starting sample: 2,87E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV231008	1/27	Homo sapiens	malignant ascites	(d)(U)C DC	Vyhlídalová Kotrbová A	2024	75%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 75% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 1 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: CD9/ CD81/ Flotillin-1/ Flotillin-2 non-EV: Apolipoprotein A-1/ Albumin/ Calreticulin/ GM130/ PMP70/ Argonaute-2/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 34 Cushion volume 4 Density of the cushion 30% Centrifugation time 70 Centrifugation speed 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ Flotillin-2 Detected contaminants Apolipoprotein A-1 Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ GM130/ PMP70/ Apolipoprotein A-1 Not detected contaminants Argonaute-2/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 188±24 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	2/27	Homo sapiens	malignant ascites	(d)(U)C UF qEV	Vyhlídalová Kotrbová A	2024	75%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 75% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 1 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: CD9/ CD81/ Flotillin-1/ Flotillin-2 non-EV: Apolipoprotein A-1/ Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Argonaute-2/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Polyethersulfone (PES) Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ Flotillin-2 Detected contaminants Apolipoprotein A-1 Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin Not detected contaminants Argonaute-2/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 689±205 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	3/27	Homo sapiens	malignant ascites	(d)(U)C DC	Vyhlídalová Kotrbová A	2024	75%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 75% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: CD9/ CD81/ Flotillin-1/ Flotillin-2 non-EV: Apolipoprotein A-1/ Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 34 Cushion volume 4 Density of the cushion 30% Centrifugation time 70 Centrifugation speed 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ Flotillin-2 Detected contaminants Apolipoprotein A-1 Proteomics database ProteomeXchange Detected contaminants Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Apolipoprotein A-1 Not detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 200±3 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	4/27	Homo sapiens	malignant ascites	(d)(U)C UF qEV	Vyhlídalová Kotrbová A	2024	75%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 75% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 2 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: CD9/ CD81/ Flotillin-1/ Flotillin-2 non-EV: Apolipoprotein A-1/ Albumin/ Calreticulin/ PMP70/ Prohibitin/ Argonaute-2/ GM130/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Polyethersulfone (PES) Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD81/ Flotillin-1/ Flotillin-2 Detected contaminants Apolipoprotein A-1 Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ PMP70/ Prohibitin/ Apolipoprotein A-1 Not detected contaminants Argonaute-2/ GM130/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 1009±417 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV240137	2/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	67%
Study summary Full title Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway All authors Luis A. Arteaga-Blanco, Andrew E. Evans, Dan A. Dixon Journal Cells Abstract NA (show more...)NA (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ actin-beta non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type S55-S Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 2 Wash: time (min) 70 Wash: Rotor Type S55-S Wash: speed (g) 150000 Filtration steps 0.8 µm/ 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD63/ CD81/ actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 108 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.10E+08 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 120

	EV240137	4/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	67%
Study summary Full title Plasma-Derived Extracellular Vesicles and Non-Extracellular Vesicle Components from APCMin/+ Mice Promote Pro-Tumorigenic Activities and Activate Human Colonic Fibroblasts via the NF-κB Signaling Pathway All authors Luis A. Arteaga-Blanco, Andrew E. Evans, Dan A. Dixon Journal Cells Abstract NA (show more...)NA (hide) EV-METRIC 67% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin APCMin/+ CRC mice model Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ actin-beta non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type S55-S Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 2 Wash: time (min) 70 Wash: Rotor Type S55-S Wash: speed (g) 150000 Filtration steps 0.8 µm/ 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Fluorometric assay Western Blot Detected EV-associated proteins CD63/ CD81/ actin-beta Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.36E+09 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 132

	EV231008	5/27	Homo sapiens	malignant ascites	(d)(U)C DC	Vyhlídalová Kotrbová A	2024	67%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 67% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 3 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: None non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Apolipoprotein A-1/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 34 Cushion volume 4 Density of the cushion 30% Centrifugation time 70 Centrifugation speed 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Detected contaminants Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Apolipoprotein A-1 Not detected contaminants Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 403±47 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	6/27	Homo sapiens	malignant ascites	(d)(U)C UF qEV	Vyhlídalová Kotrbová A	2024	67%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 67% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 3 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: None non-EV: Albumin/ Calreticulin/ PMP70/ Prohibitin/ Apolipoprotein A-1/ Argonaute-2/ GM130/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Polyethersulfone (PES) Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ PMP70/ Prohibitin/ Apolipoprotein A-1 Not detected contaminants Argonaute-2/ GM130/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 730±158 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	7/27	Homo sapiens	malignant ascites	(d)(U)C DC	Vyhlídalová Kotrbová A	2024	67%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 67% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 4 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: None non-EV: Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Apolipoprotein A-1/ Argonaute-2/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density cushion Density medium Sucrose Sample volume 34 Cushion volume 4 Density of the cushion 30% Centrifugation time 70 Centrifugation speed 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Apolipoprotein A-1 Not detected contaminants Argonaute-2/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 222±22 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	8/27	Homo sapiens	malignant ascites	(d)(U)C UF qEV	Vyhlídalová Kotrbová A	2024	67%
Study summary Full title Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer. All authors Vyhlídalová Kotrbová A, Gömöryová K, Mikulová A, Plešingerová H, Sladeček S, Kravec M, Hrachovinová Š, Potěšil D, Dunsmore G, Blériot C, Bied M, Kotouček J, Bednaříková M, Hausnerová J, Minář L, Crha I, Felsinger M, Zdráhal Z, Ginhoux F, Weinberger V, Bryja V, Pospíchalová V Journal J Extracell Vesicles Abstract High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type (show more...)High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics. (hide) EV-METRIC 67% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type malignant ascites Sample origin ovarian cancer (HGSC) patient 4 Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration qEV Protein markers EV: None non-EV: Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Apolipoprotein A-1/ Argonaute-2/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type malignant ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 10 Membrane type Polyethersulfone (PES) Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database ProteomeXchange Detected contaminants Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Apolipoprotein A-1 Not detected contaminants Argonaute-2/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean±SD Reported size (nm) 509±115 Used for determining EV concentration? Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230995	1/6	Homo sapiens	MML-1	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ Flotillin-1 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MML-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Cell count 50000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD63/ CD81/ Flotillin-1 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 761800000 EM EM-type Transmission-EM Image type Close-up

	EV230995	2/6	Homo sapiens	UM22Bap1+/+	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ Flotillin-1 non-EV: Calnexin/ bovine HBA1c/ bovine CPN1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UM22Bap1+/+ EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Cell count 50000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD63/ CD81/ Flotillin-1 Not detected contaminants Calnexin ELISA Detected contaminants bovine HBA1c/ bovine CPN1 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 936666666.7 EM EM-type Transmission-EM Image type Close-up

	EV230995	3/6	Homo sapiens	UM22Bap1-/-	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD81/ Flotillin-1/ CD63 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UM22Bap1-/- EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 93 Cell count 50000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD81/ Flotillin-1 Not detected EV-associated proteins CD63 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 36277777.78 EM EM-type Transmission-EM Image type Close-up

	EV230995	4/6	Homo sapiens	MML-1	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ Flotillin-1 non-EV: Calnexin/ bovine CPN1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MML-1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Cell count 50000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD63/ CD81/ Flotillin-1 Not detected contaminants Calnexin ELISA Detected contaminants bovine CPN1 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 139727777.8 EM EM-type Transmission-EM Image type Close-up

	EV230995	5/6	Homo sapiens	UM22Bap1+/+	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ Flotillin-1 non-EV: Calnexin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UM22Bap1+/+ EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Cell count 50000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD63/ CD81/ Flotillin-1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 82011111.11 EM EM-type Transmission-EM Image type Close-up

	EV230995	6/6	Homo sapiens	UM22Bap1-/-	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD63/ CD81/ Flotillin-1 non-EV: Calnexin/ bovine CPN1 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UM22Bap1-/- EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 93 Cell count 50000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD63/ CD81/ Flotillin-1 Not detected contaminants Calnexin ELISA Detected contaminants bovine CPN1 Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 342605555.6 EM EM-type Transmission-EM Image type Close-up

	EV230994	2/6	Bos taurus	Commercially available FBS	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Commercially available FBS Sample origin HI-before EV-depl Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: HSP70/ HSP90/ HBA1 non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Commercially available FBS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 16500 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ HSP90/ HBA1 Proteomics database No Detected contaminants Albumin/ Argonaute-2/ Calreticulin Not detected contaminants GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 32260000 EM EM-type Transmission-EM Image type Close-up

	EV230994	4/6	Bos taurus	Commercially available FBS	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Commercially available FBS Sample origin no-HI Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: HSP70/ HSP90/ HBA1 non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Commercially available FBS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ HSP90/ HBA1 Proteomics database No Detected contaminants Albumin/ Argonaute-2/ Calreticulin Not detected contaminants GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 148220000 EM EM-type Transmission-EM Image type Close-up

	EV230994	6/6	Bos taurus	Commercially available FBS	(d)(U)C	Urzì O	2024	67%
Study summary Full title Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles. All authors Urzì O, Bergqvist M, Lässer C, Moschetti M, Johansson J, D Arrigo D, Olofsson Bagge R, Crescitelli R Journal J Extracell Vesicles Abstract The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be (show more...)The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion. (hide) EV-METRIC 67% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Commercially available FBS Sample origin HI-after EV-depl Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: HSP70/ HSP90/ HBA1 non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type Commercially available FBS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 118000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ HSP90/ HBA1 Proteomics database No Detected contaminants Albumin/ Argonaute-2/ Calreticulin Not detected contaminants GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 133940000 EM EM-type Transmission-EM Image type Close-up

	EV230572	3/6	Homo sapiens	HEK293-GFP	(d)(U)C	Djeungoue-Petga, Marie-Ange	2024	67%
Study summary Full title A simple scalable extracellular vesicle isolation method using polyethylenimine polymers for use in cellular delivery All authors Marie Ange Djeungoue Petgaa, Catherine Taylora, Alexander Macpherson, Surendar Reddy Dhadi, Thomas Rollin, Jeremy W. Roya, Anirban Ghosh, Stephen M. Lewis, Rodney J. Ouellette Journal Abstract Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular (show more...)Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular delivery of therapeutic cargo. Precipitation-based methods for the isolation of EVs remain popular due to ease of use and lack of requirements for specialized equipment. We describe here a novel charge-based EV isolation method that is simple, scalable, and uses inexpensive polyethylenimine (PEI) polymers. GFP-expressing EVs were isolated from the conditioned cell culture (CCM) media of HEK293-GFP cells using either branched 10 kDa PEI (B-PEI) or linear 25 kDa PEI (L-PEI). Isolated EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy (TEM), and flow cytometry. Western blotting for common EV markers, including CD63, CD9, flotillin-1, and heat shock protein 70 were positive, while GRP94, a marker for cellular contamination, was negative. Isolated EVs had a mean diameter of 146 nm for B-PEI and 175 nm for L- PEI, while TEM revealed a spherical cup-shaped appearance typical of EVs. In addition, we determined that PEI-based EV isolation methods were scalable up to volumes of at least 50 mL. EVs isolated from CCM collected from SUM159 cells that express CD63 fused to a dual EGFP-Renilla-split tag were tested for their ability to reconstitute functional luciferase by delivering the CD63-EGFP-Renilla-split tag to SUM159 recipient cells loaded with a cytopermeable Renilla luciferase substrate. Although EVs isolated using L-PEI behaved similarly to EVs isolated using ultracentrifugation, we observed that EVs isolated using B-PEI produced a more rapid uptake and delivery of active luciferase. In this study we demonstrate that both branched and linear PEI polymers can precipitate EVs from CCM. Furthermore, once eluted from the polymers, the isolated EVs were able to deliver functional protein cargo to recipient cells. Overall, our data support PEI-based isolation of EVs as a simple, rapid method for the recovery of functional EVs. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin GFP overexpression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ Flotillin-1/ HSP70/ GFP non-EV: GRP94 Proteomics no Show all info Study aim Mechanism of uptake/transfer/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293-GFP EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ Flotillin-1/ HSP70/ GFP Not detected contaminants GRP94 Flow cytometry Type of Flow cytometry Beckman Coulter Cytoflex Hardware adaptation to ~100nm EV's The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells) Calibration bead size 0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9 Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) ~172 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.00E+09 Particle analysis: flow cytometry Flow cytometer type Nanoscale Hardware adjustment The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells) Calibration bead size 0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 9.00E+06 EM EM-type Transmission-EM Image type Close-up

	EV230029	1/2	Homo sapiens	MIA PaCa-2	(d)(U)C EX01-25L Exo-spin Standard Kit	Nannan, Lise	2024	67%
Study summary Full title Dysregulation of intercellular communication in vitro and in vivo via extracellular vesicles secreted by pancreatic duct adenocarcinoma cells and generated under the influence of the AG9 elastin peptide-conditioned microenvironment All authors Lise Nannan, Salomé Decombis, Christine Terryn, Sandra Audonnet, Jean Michel, Sylvie Brassart-Pasco, Willy Gsell, Uwe Himmelreich, Bertrand Brassart Journal J Extracell Biol Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis due to its h (show more...)Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis due to its highly metastatic profile. Intercellular communication between cancer and stromal cells via extracellular vesicles (EVs) is crucial for the premetastatic microenvironment preparation leading to tumour metastasis. This study shows that under the influence of bioactive peptides derived from the extracellular matrix microenvironment, illustrated here by the AG-9 elastin-derived peptide (EDP), PDAC cells secrete more tumour-derived EVs. Compared to PDAC-derived EVs, tumour-derived EVs resulting from AG-9 treatment (PDAC AG-9-derived EVs) significantly stimulated cell proliferation. At constant amount, tumour-derived EVs were similarly taken up by PDAC and HMEC-1 cells. Tumour-derived EVs stimulated cell proliferation, migration, proteinase secretion, and angiogenesis. Bioluminescence imaging allowed tumour-derived EV/FLuc+ tracking in vivo in a PDAC mouse model. The biodistribution of PDAC AG-9-derived EVs was different to PDAC-derived EVs. Our results demonstrate that the microenvironment, through EDP release, may not only influence the genesis of EVs but may also affect tumour progression (tumour growth and angiogenesis), and metastatic homing by modifying the in vivo biodistribution of tumour-derived EVs. They are potential candidates for targeted drug delivery and modulation of tumour progression, and they constitute a new generation of therapeutic tools, merging oncology and genic therapy. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin eGFP/FLuc overexpression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD63/ CD81 non-EV: GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 97 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 0.1 Wash: time (min) 70 Wash: Rotor Type TLA-100.4 Wash: speed (g) 100000 Commercial kit EX01-25L Exo-spin Standard Kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants GM130 Flow cytometry aspecific beads Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Selected surface protein(s) CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment Yes RNAse type RNase A RNAse concentration 0.004 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+08 EM EM-type Transmission-EM/ Immuno-EM EM protein CD63 Image type Close-up Report size (nm) 120

	EV230029	2/2	Homo sapiens	MIA PaCa-2	(d)(U)C EX01-25L Exo-spin Standard Kit	Nannan, Lise	2024	67%
Study summary Full title Dysregulation of intercellular communication in vitro and in vivo via extracellular vesicles secreted by pancreatic duct adenocarcinoma cells and generated under the influence of the AG9 elastin peptide-conditioned microenvironment All authors Lise Nannan, Salomé Decombis, Christine Terryn, Sandra Audonnet, Jean Michel, Sylvie Brassart-Pasco, Willy Gsell, Uwe Himmelreich, Bertrand Brassart Journal J Extracell Biol Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis due to its h (show more...)Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis due to its highly metastatic profile. Intercellular communication between cancer and stromal cells via extracellular vesicles (EVs) is crucial for the premetastatic microenvironment preparation leading to tumour metastasis. This study shows that under the influence of bioactive peptides derived from the extracellular matrix microenvironment, illustrated here by the AG-9 elastin-derived peptide (EDP), PDAC cells secrete more tumour-derived EVs. Compared to PDAC-derived EVs, tumour-derived EVs resulting from AG-9 treatment (PDAC AG-9-derived EVs) significantly stimulated cell proliferation. At constant amount, tumour-derived EVs were similarly taken up by PDAC and HMEC-1 cells. Tumour-derived EVs stimulated cell proliferation, migration, proteinase secretion, and angiogenesis. Bioluminescence imaging allowed tumour-derived EV/FLuc+ tracking in vivo in a PDAC mouse model. The biodistribution of PDAC AG-9-derived EVs was different to PDAC-derived EVs. Our results demonstrate that the microenvironment, through EDP release, may not only influence the genesis of EVs but may also affect tumour progression (tumour growth and angiogenesis), and metastatic homing by modifying the in vivo biodistribution of tumour-derived EVs. They are potential candidates for targeted drug delivery and modulation of tumour progression, and they constitute a new generation of therapeutic tools, merging oncology and genic therapy. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin eGFP/FLuc overexpression, AG-9 treated Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD9/ CD63/ CD81 non-EV: GM130 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MIA PaCa-2 EV-harvesting Medium Serum free medium Cell viability (%) 97 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 0.1 Wash: time (min) 70 Wash: Rotor Type TLA-100.4 Wash: speed (g) 100000 Commercial kit EX01-25L Exo-spin Standard Kit Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Not detected contaminants GM130 Flow cytometry aspecific beads Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Selected surface protein(s) CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment Yes RNAse type RNase A RNAse concentration 0.004 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 120 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+08 EM EM-type Transmission-EM Image type Close-up Report size (nm) 120

	EV240036	5/6	Homo sapiens	Serum	Exoquick IAF	Shinde U	2024	63%
Study summary Full title Isolation of serum-derived placental/amnio-chorionic extracellular vesicles across pregnancy by immunoaffinity using PLAP and HLA-G. All authors Shinde U, Rao A, Bansal V, Das DK, Balasinor NH, Madan T Journal Reproduction Abstract Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic (show more...)Circulating extracellular vesicles of placental/amniochorionic origin carry placental/amniochorionic proteins and nucleic acids with the potential to facilitate non-invasive diagnosis of pregnancy-related disorders. The study reports an improvised method for the enriched isolation of extracellular vesicles of placental/amniochorionic origin using the two markers, PLAP and HLA-G. (hide) EV-METRIC 63% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Pregnant Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Commercial method Immunoaffinity capture (non-commercial) Protein markers EV: CD9/ CD63/ PLAP/ Cullin 7 non-EV: Calnexin Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit Exoquick Immunoaffinity capture Selected surface protein(s) PLAP EV-subtype Distinction between multiple subtypes affinity capture Used subtypes PLAP+ Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD63/ PLAP/ Cullin 7 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type RT(q)PCR RNAse treatment Yes Moment of RNAse treatment After RNAse type RNAse RNAse concentration 20mg/mL Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 30-150 nm NTA Report type Modus Reported size (nm) 30-150 nm EV concentration Yes Particle yield number of particles per million cells: 1E10 particles/ml EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-150 nm

	EV230572	4/6	Homo sapiens	HEK293-GFP	PEI precipitation	Djeungoue-Petga, Marie-Ange	2024	63%
Study summary Full title A simple scalable extracellular vesicle isolation method using polyethylenimine polymers for use in cellular delivery All authors Marie Ange Djeungoue Petgaa, Catherine Taylora, Alexander Macpherson, Surendar Reddy Dhadi, Thomas Rollin, Jeremy W. Roya, Anirban Ghosh, Stephen M. Lewis, Rodney J. Ouellette Journal Abstract Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular (show more...)Extracellular vesicles (EVs) are gaining interest as efficient, biocompatible vehicles for cellular delivery of therapeutic cargo. Precipitation-based methods for the isolation of EVs remain popular due to ease of use and lack of requirements for specialized equipment. We describe here a novel charge-based EV isolation method that is simple, scalable, and uses inexpensive polyethylenimine (PEI) polymers. GFP-expressing EVs were isolated from the conditioned cell culture (CCM) media of HEK293-GFP cells using either branched 10 kDa PEI (B-PEI) or linear 25 kDa PEI (L-PEI). Isolated EVs were characterized by Western blotting, nanoparticle tracking analysis, transmission electron microscopy (TEM), and flow cytometry. Western blotting for common EV markers, including CD63, CD9, flotillin-1, and heat shock protein 70 were positive, while GRP94, a marker for cellular contamination, was negative. Isolated EVs had a mean diameter of 146 nm for B-PEI and 175 nm for L- PEI, while TEM revealed a spherical cup-shaped appearance typical of EVs. In addition, we determined that PEI-based EV isolation methods were scalable up to volumes of at least 50 mL. EVs isolated from CCM collected from SUM159 cells that express CD63 fused to a dual EGFP-Renilla-split tag were tested for their ability to reconstitute functional luciferase by delivering the CD63-EGFP-Renilla-split tag to SUM159 recipient cells loaded with a cytopermeable Renilla luciferase substrate. Although EVs isolated using L-PEI behaved similarly to EVs isolated using ultracentrifugation, we observed that EVs isolated using B-PEI produced a more rapid uptake and delivery of active luciferase. In this study we demonstrate that both branched and linear PEI polymers can precipitate EVs from CCM. Furthermore, once eluted from the polymers, the isolated EVs were able to deliver functional protein cargo to recipient cells. Overall, our data support PEI-based isolation of EVs as a simple, rapid method for the recovery of functional EVs. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin GFP overexpresion Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture PEI precipitation Protein markers EV: CD9/ CD63/ Flotillin-1/ HSP70/ GFP non-EV: GRP94 Proteomics no Show all info Study aim Mechanism of uptake/transfer/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293-GFP EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Other Name other separation method PEI precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ Flotillin-1/ HSP70/ GFP Not detected contaminants GRP94 Flow cytometry Type of Flow cytometry Beckman Coulter Cytoflex Hardware adaptation to ~100nm EV's The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells) Calibration bead size 0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9 Detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 146-200 EV concentration Yes Particle yield particles per milliliter of starting sample: 1-6E10 Particle analysis: flow cytometry Flow cytometer type Nanoscale Hardware adjustment The better resolution of the CytoFLEX is reached by using the violet side scatter of the 405 nm laser (manually set to 1600 and height threshold) and by performing preanalytical preparations with Fluorescent Megamix-Plus SSC beads (Cosmo Bio Co., LTD, Japan) which are FITC-labeled beads of increasing size (100, 160, 200, 240, 300, 500, 900 nm). beads were used to set the EV gate and manual gating was set to the populations of interest with reference to a negative control sample (GFP- EVs from HEK293 cells) Calibration bead size 0.1/ 0.16/ 0.2/ 0.24/ 0.3/ 0.5/ 0.9 Particle yield particles per milliliter of starting sample: 1-6E10 EM EM-type Transmission-EM Image type Close-up

	EV230012	1/4	Mus musculus	cardiac mesenchymal stromal cells	Filtration UF SEC (non-commercial)	Caller T	2024	63%
Study summary Full title Small Extracellular Vesicles From Infarcted and Failing Heart Accelerate Tumor Growth. All authors Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J Journal Circulation Abstract Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD81/ TSG101 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells cardiac mesenchymal stromal cells EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 3000000 Separation Method Filtration steps 0.8 Ultra filtration Cut-off size (kDa) 10 Membrane type Cellulose acetate Size-exclusion chromatography Total column volume (mL) 70 Sample volume/column (mL) 10 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) >250 EV concentration Yes Particle yield number of particles per million cells: 90000000 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230012	2/4	Mus musculus	cardiac mesenchymal stromal cells	Filtration UF SEC (non-commercial)	Caller T	2024	63%
Study summary Full title Small Extracellular Vesicles From Infarcted and Failing Heart Accelerate Tumor Growth. All authors Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J Journal Circulation Abstract Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Heart failure Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD81/ TSG101 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells cardiac mesenchymal stromal cells EV-harvesting Medium Serum free medium Cell viability (%) 90 Cell count 3000000 Separation Method Filtration steps 0.8 Ultra filtration Cut-off size (kDa) 10 Membrane type Cellulose acetate Size-exclusion chromatography Total column volume (mL) 70 Sample volume/column (mL) 10 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) <250 EV concentration Yes Particle yield number of particles per million cells: 40000000 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230012	3/4	Mus musculus	Cardiac tissue	Filtration UF SEC (non-commercial)	Caller T	2024	63%
Study summary Full title Small Extracellular Vesicles From Infarcted and Failing Heart Accelerate Tumor Growth. All authors Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J Journal Circulation Abstract Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide) EV-METRIC 63% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cardiac tissue Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD81/ TSG101 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cardiac tissue Separation Method Filtration steps 0.8 Ultra filtration Cut-off size (kDa) 10 Membrane type Cellulose acetate Size-exclusion chromatography Total column volume (mL) 70 Sample volume/column (mL) 10 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) <250 EV concentration Yes Particle yield number of particles per 100mg tissue: 90000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230012	4/4	Mus musculus	Cardiac tissue	Filtration UF SEC (non-commercial)	Caller T	2024	63%
Study summary Full title Small Extracellular Vesicles From Infarcted and Failing Heart Accelerate Tumor Growth. All authors Caller T, Rotem I, Shaihov-Teper O, Lendengolts D, Schary Y, Shai R, Glick-Saar E, Dominissini D, Motiei M, Katzir I, Popovtzer R, Nahmoud M, Boomgarden A, D'Souza-Schorey C, Naftali-Shani N, Leor J Journal Circulation Abstract Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. H (show more...)Myocardial infarction (MI) and heart failure are associated with an increased incidence of cancer. However, the mechanism is complex and unclear. Here, we aimed to test our hypothesis that cardiac small extracellular vesicles (sEVs), particularly cardiac mesenchymal stromal cell-derived sEVs (cMSC-sEVs), contribute to the link between post-MI left ventricular dysfunction (LVD) and cancer. (hide) EV-METRIC 63% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cardiac tissue Sample origin Heart failure Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD81/ TSG101 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cardiac tissue Separation Method Filtration steps 0.8 Ultra filtration Cut-off size (kDa) 10 Membrane type Cellulose acetate Size-exclusion chromatography Total column volume (mL) 70 Sample volume/column (mL) 10 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) <250 EV concentration Yes Particle yield number of particles per 100 mg tissue: 190000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220300	7/18	Homo sapiens	CT5.3hTeRT	UF (d)(U)C Filtration DG SEC (non-commercial)	Pinheiro C	2024	63%
Study summary Full title Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data. All authors Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, Van Dorpe S, De Craene B, Berx G, Boterberg T, Sys G, Denys H, Miinalainen I, Mestdagh P, Vandesompele J, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diver (show more...)Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration (Differential) (ultra)centrifugation Filtration Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ TSG101/ Flotillin-1/ Syntenin-1/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Validation of standards Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CT5.3hTeRT EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 15.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 _m Ultra filtration Cut-off size (kDa) 10 kDa Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) particles per milliliter of starting sample: 2.10E10-8.93E10 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 139.6-150.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.10E10-8.93E10

	EV220300	8/18	Homo sapiens	CT5.3hTeRT	UF (d)(U)C Filtration DG SEC (non-commercial)	Pinheiro C	2024	63%
Study summary Full title Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data. All authors Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, Van Dorpe S, De Craene B, Berx G, Boterberg T, Sys G, Denys H, Miinalainen I, Mestdagh P, Vandesompele J, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diver (show more...)Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 10Gy single dose Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration (Differential) (ultra)centrifugation Filtration Density gradient Size-exclusion chromatography (non-commercial) Protein markers EV: Alix/ TSG101/ Flotillin-1/ Syntenin-1/ CD9 non-EV: None Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Validation of standards Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells CT5.3hTeRT EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 15.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 _m Ultra filtration Cut-off size (kDa) 10 kDa Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Not determined Protein Yield (µg) particles per milliliter of starting sample: 2.10E10-8.93E10 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 139.6-150.4 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.10E10-8.93E10

	EV210336	1/2	Homo sapiens	THP1	(d)(U)C DG	Driedonks, Tom	2024	63%
Study summary Full title Intracellular localisation and extracellular release of Y RNA and Y RNA binding proteins All authors Tom A. P. Driedonks, Sarah Ressel, Thi Tran Ngoc Minh, Amy H. Buck, Esther N. M. Nolte-‘t Hoen Journal J Extracell Biol Abstract Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-siz (show more...)Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.11 -1.18 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4 M Highest density fraction 2.0 M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.2 Orientation Bottom-up Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: speed (g) 192000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment according to van der Vlist et al, Nature Protocols 2012 Calibration bead size 0.1/ 0.2 EV concentration Yes

	EV210336	2/2	Homo sapiens	THP1	(d)(U)C DG	Driedonks, Tom	2024	63%
Study summary Full title Intracellular localisation and extracellular release of Y RNA and Y RNA binding proteins All authors Tom A. P. Driedonks, Sarah Ressel, Thi Tran Ngoc Minh, Amy H. Buck, Esther N. M. Nolte-‘t Hoen Journal J Extracell Biol Abstract Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-siz (show more...)Cells can communicate via the release and uptake of extracellular vesicles (EVs), which are nano-sized membrane vesicles that can transfer protein and RNA cargo between cells. EVs contain microRNAs and various other types of non-coding RNA, of which Y RNA is among the most abundant types. Studies on how RNAs and their binding proteins are sorted into EVs have mainly focused on comparing intracellular (cytoplasmic) levels of these RNAs to the extracellular levels in EVs. Besides overall transcriptional levels that may regulate sorting of RNAs into EVs, the process may also be driven by local intracellular changes in RNA/RBP concentrations. Changes in extracellular Y RNA have been linked to cancer and cardiovascular diseases. Although the loading of RNA cargo into EVs is generally thought to be influenced by cellular stimuli and regulated by RNA binding proteins (RBP), little is known about Y RNA shuttling into EVs. We previously reported that immune stimulation alters the levels of Y RNA in EVs independently of cytosolic Y RNA levels. This suggests that Y RNA binding proteins, and/or changes in the local Y RNA concentration at EV biogenesis sites, may affect Y RNA incorporation into EVs. Here, we investigated the subcellular distribution of Y RNA and Y RNA binding proteins in activated and non-activated THP1 macrophages. We demonstrate that Y RNA and its main binding protein Ro60 abundantly co-fractionate in organelles involved in EV biogenesis and in EVs. Cellular activation led to an increase in Y RNA concentration at EV biogenesis sites and this correlated with increased EV-associated levels of Y RNA and Ro60. These results suggest that Y RNA incorporation into EVs may be controlled by local intracellular changes in the concentration of Y RNA and their protein binding partners. (hide) EV-METRIC 63% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Pam3CSK4 treated Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.11 -1.18 Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells THP1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 95 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Continuous Lowest density fraction 0.4 M Highest density fraction 2.0 M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 0.2 Orientation Bottom-up Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 4 Pelleting: speed (g) 192000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD Influx Hardware adjustment according to van der Vlist et al Nature Protocols 2012 Calibration bead size 0.1/ 0.2 EV concentration Yes

	EV220300	12/18	Homo sapiens	PANC1	UF (d)(U)C Filtration DG	Pinheiro C	2024	57%
Study summary Full title Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data. All authors Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, Van Dorpe S, De Craene B, Berx G, Boterberg T, Sys G, Denys H, Miinalainen I, Mestdagh P, Vandesompele J, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diver (show more...)Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Validation of standards Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells PANC1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 92 Cell count 2.06e8 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW32.1 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 15.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 _m Ultra filtration Cut-off size (kDa) 10 kDa Membrane type Regenerated cellulose Size-exclusion chromatography Resin type EV-subtype Distinction between multiple subtypes Size Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Protein Yield (µg) per microliter Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-90

	EV220300	13/18	Homo sapiens	T47D	UF (d)(U)C Filtration DG	Pinheiro C	2024	57%
Study summary Full title Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data. All authors Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, Van Dorpe S, De Craene B, Berx G, Boterberg T, Sys G, Denys H, Miinalainen I, Mestdagh P, Vandesompele J, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diver (show more...)Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Validation of standards Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells T47D EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW32.1 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 15.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 _m Ultra filtration Cut-off size (kDa) 10 kDa Membrane type Regenerated cellulose Size-exclusion chromatography Resin type Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Protein Yield (µg) per microliter Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 90-120

	EV220300	14/18	Homo sapiens	U87MG	UF (d)(U)C Filtration DG	Pinheiro C	2024	57%
Study summary Full title Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data. All authors Pinheiro C, Guilbert N, Lippens L, Roux Q, Boiy R, Fischer S, Van Dorpe S, De Craene B, Berx G, Boterberg T, Sys G, Denys H, Miinalainen I, Mestdagh P, Vandesompele J, De Wever O, Hendrix A Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diver (show more...)Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture Ultrafiltration (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.09-1.11 Show all info Study aim Validation of standards Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U87MG EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 94 Cell count 6.52e8 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW32.1 Ti Pelleting: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 15.5 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 16 Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 _m Ultra filtration Cut-off size (kDa) 10 kDa Membrane type Regenerated cellulose Size-exclusion chromatography Resin type Characterization: Protein analysis None Protein Concentration Method Fluorometric assay Protein Yield (µg) per microliter Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 120-250

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