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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	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

	EV240137	1/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	56%
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 56% (87th 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 large 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 Pelleting performed Yes Pelleting: speed (g) 15000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 40 Wash: speed (g) 15000 Filtration steps 0.8 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) 191 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.29E+08 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 246 Extra information This study characterizes plasma-derived EV subtypes (large and small EVs) and other non-vesicular entities released from the colorectal (CRC) mouse model APCMin/+. EV preparations, including large EVs (LEV) and small EVs (SEV), were characterized based on their physical (NTA and SEM) and biochemical properties (WB). Because we did not use additional purification methodologies such as size exclusion chromatography, density gradient, immunocapture, etc., we expected our crude EV preparations to be contaminated with other extracellular particles (e.g., lipoproteins) and soluble proteins such as albumin, immunoglobulins, and fibrinogen. Therefore, the operational terms EV preparation or EV-containing preparations were used to refer to nanoparticles obtained by multiple centrifugation steps containing populations of LEV or SEV and non-vesicular entities. We believe this is an excellent opportunity to share our protocol for reproducibility purposes with other researchers working with plasma-

	EV240137	3/4	Mus musculus	Blood plasma	(d)(U)C Filtration	Arteaga-Blanco, Luis A.	2024	56%
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 56% (87th 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 large 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 Pelleting performed Yes Pelleting: speed (g) 15000 Wash: volume per pellet (ml) 1.4 Wash: time (min) 40 Wash: speed (g) 15000 Filtration steps 0.8 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) 246 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.41E+09 EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 290

	EV240017	1/3	Ceratotherium simum simum	Follicular Fluid	(d)(U)C Filtration SEC (non-commercial)	Gad A	2024	56%
Study summary Full title Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum). All authors Gad A, Menjivar NG, Felton R, Durrant B, Tesfaye D, Ruggeri E Journal Biol Reprod Abstract Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the (show more...)Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR/ Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR/ Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G/ 11-17 mm), Dominant (D/ 18-29 mm), and Pre-ovulatory (P/ 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNA previously known via subsequent annotation to all equine (Equus caballus/ eca), bovine (Bos taurus/ bta), and human (Homo sapiens/ hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts. (hide) EV-METRIC 56% (76th 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 Follicular Fluid Sample origin Growing follicle 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 Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: HSP70/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Ceratotherium simum simum Sample Type Follicular Fluid 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 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0.5 Sample volume/column (mL) 0.75 Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 173.78 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.93E+09 EM EM-type Transmission-EM Image type Close-up

	EV240017	2/3	Ceratotherium simum simum	Follicular Fluid	(d)(U)C Filtration SEC (non-commercial)	Gad A	2024	56%
Study summary Full title Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum). All authors Gad A, Menjivar NG, Felton R, Durrant B, Tesfaye D, Ruggeri E Journal Biol Reprod Abstract Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the (show more...)Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR/ Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR/ Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G/ 11-17 mm), Dominant (D/ 18-29 mm), and Pre-ovulatory (P/ 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNA previously known via subsequent annotation to all equine (Equus caballus/ eca), bovine (Bos taurus/ bta), and human (Homo sapiens/ hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts. (hide) EV-METRIC 56% (76th 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 Follicular Fluid Sample origin Dominant follicle 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 Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: HSP70/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Ceratotherium simum simum Sample Type Follicular Fluid 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 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0.5 Sample volume/column (mL) 0.75 Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 225.76 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.17E+09 EM EM-type Transmission-EM Image type Close-up

	EV240017	3/3	Ceratotherium simum simum	Follicular Fluid	(d)(U)C Filtration SEC (non-commercial)	Gad A	2024	56%
Study summary Full title Mapping the follicle-specific regulation of extracellular vesicle-mediated microRNA transport in the southern white rhinoceros (Ceratotherium simum simum). All authors Gad A, Menjivar NG, Felton R, Durrant B, Tesfaye D, Ruggeri E Journal Biol Reprod Abstract Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the (show more...)Efforts to implement effective assisted reproductive technologies (ARTs) for the conservation of the northern white rhinoceros (NWR/ Ceratotherium simum cottoni) to prevent its forthcoming extinction, could be supported by research conducted on the closely related southern white rhinoceros (SWR/ Ceratotherium simum simum). Within the follicle, extracellular vesicles (EVs) play a fundamental role in the bidirectional communication facilitating the crucial transport of regulatory molecules such as microRNAs (miRNAs) that control follicular growth and oocyte development. This study aimed to elucidate the dynamics of EV-miRNAs in stage-dependent follicular fluid (FF) during SWR ovarian antral follicle development. Three distinct follicular stages were identified based on diameter: Growing (G/ 11-17 mm), Dominant (D/ 18-29 mm), and Pre-ovulatory (P/ 30-34 mm). Isolated EVs from the aspirated FF of segmented follicle stages were used to identify EV-miRNA previously known via subsequent annotation to all equine (Equus caballus/ eca), bovine (Bos taurus/ bta), and human (Homo sapiens/ hsa) miRNAs. A total of 417 miRNAs were detected, with 231 being mutually expressed across all three stages, including eca-miR-148a and bta-miR-451 as the top highly expressed miRNAs. Distinct expression dynamics in miRNA abundance were observed across the three follicular stages, including 31 differentially expressed miRNAs that target various pathways related to follicular growth and development, with 13 miRNAs commonly appearing amidst two different comparisons. In conclusion, this pioneering study provides a comprehensive understanding of the stage-specific expression dynamics of FF EV-miRNAs in the SWR. These findings provide insights that may lead to novel approaches in enhancing ARTs to catalyze rhinoceros conservation efforts. (hide) EV-METRIC 56% (76th 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 Follicular Fluid Sample origin Pre-ovulatory follicle 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 Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: HSP70/ TSG101 non-EV: Cytochrome C Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Ceratotherium simum simum Sample Type Follicular Fluid 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 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 0.5 Sample volume/column (mL) 0.75 Characterization: Protein analysis Protein Concentration Method Nanodrop Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins HSP70/ TSG101 Not detected contaminants Cytochrome C Characterization: RNA analysis RNA analysis Type RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 237.41 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.05E+09 EM EM-type Transmission-EM Image type Close-up

	EV230598	2/3	Homo sapiens	Milk	(d)(U)C	Ten-Doménech, Isabel	2024	56%
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 56% (76th 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 term 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 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

	EV230597	3/9	Homo sapiens	293T	(d)(U)C	Chen Z	2024	56%
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 56% (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 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: Alix/ CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells 293T EV-harvesting Medium Serum free medium Cell count 1.00E+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 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 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: 1.20E+08

	EV230597	4/9	Homo sapiens	huMSC	(d)(U)C	Chen Z	2024	56%
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 56% (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 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: CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells huMSC EV-harvesting Medium Serum free medium Cell count 5.00E+06 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 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins 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 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: 7.71E+07

	EV230597	5/9	Homo sapiens	HFF-1	(d)(U)C	Chen Z	2024	56%
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 56% (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 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: Alix/ CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HFF-1 EV-harvesting Medium Serum free medium Cell count 1.00E+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 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 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: 8.57E+07

	EV230597	6/9	Homo sapiens	A549	(d)(U)C	Chen Z	2024	56%
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 56% (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 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: Alix/ CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells A549 EV-harvesting Medium Serum free medium Cell count 1.00E+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 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 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: 9.78E+07

	EV230597	7/9	Homo sapiens	H460	(d)(U)C	Chen Z	2024	56%
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 56% (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 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: Alix/ CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells H460 EV-harvesting Medium Serum free medium Cell count 1.00E+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 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 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: 1.44E+08

	EV230597	8/9	Homo sapiens	MCF7	(d)(U)C	Chen Z	2024	56%
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 56% (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 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: Alix/ CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium Serum free medium Cell count 1.00E+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 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 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: 1.86E+08

	EV230597	9/9	Homo sapiens	MDAMB231	(d)(U)C	Chen Z	2024	56%
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 56% (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 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: Alix/ CD9/ CD63/ TSG101/ CD81 non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium Serum free medium Cell count 1.00E+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 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 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: 1.68E+08

	EV230572	1/6	Homo sapiens	HEK293	(d)(U)C	Djeungoue-Petga, Marie-Ange	2024	56%
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 56% (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 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: 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 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 Not detected EV-associated proteins 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 Not detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis None Extra information two kinds of PEI were used. Linear PEI 25 kDa and Branched 10 kDa

	EV230372	1/14	Homo sapiens	A549	(d)(U)C	Schöne N	2024	56%
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 56% (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 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 Protein markers EV: Actinin-4/ CD81/ Rgap1/ Syntenin/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin/ Arf6/ HDAC1 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 A549 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 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/ Mitofilin/ Arf6 Not detected EV-associated proteins CD81/ Rgap1/ Syntenin Detected contaminants ApoB Not detected contaminants GM130/ HDAC1/ Albumin/ 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) 163.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.71E+08

	EV230372	2/14	Homo sapiens	A549	(d)(U)C	Schöne N	2024	56%
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 56% (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 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 A549 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 CD81/ Syntenin/ Actinin-4/ Arf6 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) 147.13 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.95E+09

	EV230372	4/14	Homo sapiens	HCC-78	(d)(U)C	Schöne N	2024	56%
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 56% (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 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 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 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 CD81/ Syntenin/ Arf6 Not detected EV-associated proteins Rgap1/ Actinin-4/ Mitofilin Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 163.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.26E+10

	EV230372	5/14	Homo sapiens	H1975	(d)(U)C	Schöne N	2024	56%
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 56% (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 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 Protein markers EV: Actinin-4/ Rgap1/ Syntenin-1/ CD81/ EMMPRIN/ EpCAM/ EGFR/ Mitofilin,Arf6 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 H1975 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 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 CD81/ Syntenin Detected contaminants GM130 Not detected contaminants 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) 180.6 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.37E+10

	EV230372	6/14	Homo sapiens	H1975	(d)(U)C	Schöne N	2024	56%
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 56% (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 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 H1975 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 CD81/ Syntenin/ Actinin-4 Not detected EV-associated proteins Rgap1/ Mitofilin/ Arf6 Not detected contaminants GM130/ HDAC1/ Albumin/ ApoA1/ ApoB Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 163.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.43E+10

	EV230372	11/14	Homo sapiens	Blood plasma	(d)(U)C	Schöne N	2024	56%
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 56% (87th 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 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 non-EV: ApoA1/ ApoB Proteomics no 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 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/ ApoA1 Flow cytometry Type of Flow cytometry standard flow cytometer Calibration bead size 0.2, 0.8 Detected EV-associated proteins EMMPRIN/ EpCAM Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 185.2 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.01E+08

	EV220329	1/2	Homo sapiens	Blood plasma	(d)(U)C	Chong MC	2024	56%
Study summary Full title Acute exercise-induced release of innate immune proteins via small extracellular vesicles changes with aerobic fitness and age. All authors Chong MC, Shah AD, Schittenhelm RB, Silva A, James PF, Wu SSX, Howitt J Journal Acta Physiol (Oxf) Abstract Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation (show more...)Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation in humans, enabling signalling crosstalk between tissues. Exercise-derived EVs and their cargo have been proposed to mediate adaptations to exercise/ however, our understanding of how exercise-derived EV protein cargo is modulated by factors such as aerobic fitness and age of an individual is currently unknown. Here, we examined the circulating sEV proteome following aerobic exercise in healthy males of different ages and aerobic fitness to understand exercise-induced EV response during the aging process. (hide) EV-METRIC 56% (87th 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 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: TSG101/ Syntenin-1 non-EV: ApoA-1/ GM130 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma 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 70 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 20 Wash: time (min) 60 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins TSG101/ Syntenin-1 Detected contaminants ApoA-1 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis Other particle analysis name(1) Microfluidic resistive pulse sensing Report type Mean Report size 88 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.80E+10

	EV220329	2/2	Homo sapiens	Blood plasma	(d)(U)C	Chong MC	2024	56%
Study summary Full title Acute exercise-induced release of innate immune proteins via small extracellular vesicles changes with aerobic fitness and age. All authors Chong MC, Shah AD, Schittenhelm RB, Silva A, James PF, Wu SSX, Howitt J Journal Acta Physiol (Oxf) Abstract Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation (show more...)Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation in humans, enabling signalling crosstalk between tissues. Exercise-derived EVs and their cargo have been proposed to mediate adaptations to exercise/ however, our understanding of how exercise-derived EV protein cargo is modulated by factors such as aerobic fitness and age of an individual is currently unknown. Here, we examined the circulating sEV proteome following aerobic exercise in healthy males of different ages and aerobic fitness to understand exercise-induced EV response during the aging process. (hide) EV-METRIC 56% (87th 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 post-exercise 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: TSG101/ Syntenin-1 non-EV: ApoA-1/ GM130 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma 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 70 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 20 Wash: time (min) 60 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins TSG101/ Syntenin-1 Detected contaminants ApoA-1 Not detected contaminants GM130 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis Other particle analysis name(1) Microfluidic resistive pulse sensing Report type Mean Report size 89 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 3.90E+10

	EV240036	2/6	Homo sapiens	Serum	Exoquick IAF	Shinde U	2024	50%
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 50% (89th 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 Non 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: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 30-150 nm EV concentration Yes Particle yield number of particles per million cells: 1E10 particles/ml

	EV240036	4/6	Homo sapiens	Serum	Exoquick	Shinde U	2024	50%
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 50% (89th 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 Protein markers EV: CD9/ CD63/ PLAP/ Cullin 7 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit Exoquick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ CD63/ PLAP/ Cullin 7 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 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 30-150 nm

	EV240036	6/6	Homo sapiens	Serum	Exoquick IAF	Shinde U	2024	50%
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 50% (89th 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: PLAP/ HLA-G 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/ HLA-G EV-subtype Distinction between multiple subtypes affinity capture Used subtypes PLAP+ HLA-G+ Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins PLAP/ HLA-G Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis 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

	EV240006	3/4	Rattus norvegicus	INS-1	(d)(U)C Total Exosome Isolation lipid-based affinity capture	Weerakkody, Jonathan S.	2024	50%
Study summary Full title Photosensitive Nanoprobes for Rapid High Purity Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations All authors Jonathan S. Weerakkody, Tiffany Tseng, Mackenzie Topper, Sikha Thoduvayil, Abhijith Radhakrishnan, Frederic Pincet, Themis R. Kyriakides, Roshan W. Gunasekara, Sathish Ramakrishnan Journal Abstract The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as (show more...)The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. (hide) EV-METRIC 50% (87th 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 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 Total Exosome Isolation lipid-based affinity capture Protein markers EV: CD9/ CD81 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells INS-1 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 12500 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Other Name other separation method lipid-based affinity capture EV-subtype Distinction between multiple subtypes Size Used subtypes 30-200 Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 104 NTA Report type Mean Reported size (nm) 126 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(1) dSTORM single molecule localization microscopy Report type Mean Report size 89 EV-concentration No Extra information This paper was to validate the efficacy of photosensitive lipid nanoprobe for the isolation and size selective enrichment of native extracellular vesicles

	EV240006	4/4	Mus musculus	Neuro-2a (N2a) CCL-131	(d)(U)C Total Exosome Isolation lipid-based affinity capture	Weerakkody, Jonathan S.	2024	50%
Study summary Full title Photosensitive Nanoprobes for Rapid High Purity Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations All authors Jonathan S. Weerakkody, Tiffany Tseng, Mackenzie Topper, Sikha Thoduvayil, Abhijith Radhakrishnan, Frederic Pincet, Themis R. Kyriakides, Roshan W. Gunasekara, Sathish Ramakrishnan Journal Abstract The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as (show more...)The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. (hide) EV-METRIC 50% (87th 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 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 Total Exosome Isolation lipid-based affinity capture Protein markers EV: CD9/ CD81 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Neuro-2a (N2a) CCL-131 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 12500 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Other Name other separation method lipid-based affinity capture EV-subtype Distinction between multiple subtypes Size Used subtypes 150-350 Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 105 NTA Report type Mean Reported size (nm) 122 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.00E+07 EM EM-type Transmission-EM Image type Close-up, Wide-field Other particle analysis name(1) dSTORM single molecule localization microscopy Report type Mean Report size 52 EV-concentration No Extra information This paper was to validate the efficacy of photosensitive lipid nanoprobe for the isolation and size selective enrichment of native extracellular vesicles

	EV231008	9/27	Homo sapiens	malignant ascites	(d)(U)C DC	Vyhlídalová Kotrbová A	2024	50%
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 50% (56th 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 5 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 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	10/27	Homo sapiens	malignant ascites	(d)(U)C UF qEV	Vyhlídalová Kotrbová A	2024	50%
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 50% (56th 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 5 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 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231008	11/27	Homo sapiens	malignant ascites	(d)(U)C DC	Vyhlídalová Kotrbová A	2024	50%
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 50% (56th 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 6 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 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV231005	2/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin First 10 mL of sperm rich fraction Focus vesicles large extracellular vesicles 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal 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 24 x 1.5/2.0mL Pelleting: speed (g) 20000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 235 EM EM-type Transmission-EM Image type Wide-field

	EV231005	4/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin Remaining sperm rich fraction Focus vesicles large extracellular vesicles 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal 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 24 x 1.5/2.0mL Pelleting: speed (g) 20000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 244 EM EM-type Transmission-EM Image type Wide-field

	EV231005	6/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin Post sperm rich fraction Focus vesicles large extracellular vesicles 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal 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 24 x 1.5/2.0mL Pelleting: speed (g) 20000 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method microBCA Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 267 EM EM-type Transmission-EM Image type Wide-field

	EV231005	7/8	Sus scrofa domesticus	Seminal plasma	(d)(U)C Filtration UF SEC (non-commercial)	Barranco I	2024	50%
Study summary Full title Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism. All authors Barranco I, Spinaci M, Nesci S, Mateo-Otero Y, Baldassarro VA, Algieri C, Bucci D, Roca J Journal Theriogenology Abstract Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEV (show more...)Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry/ albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. (hide) EV-METRIC 50% (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 Seminal plasma Sample origin Entire ejaculate Focus vesicles small extracellular vesicles 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 Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81/ HSP90/ CD44 non-EV: Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Sus scrofa domesticus Sample Type Seminal 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 No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 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 microBCA Western Blot Not detected contaminants Albumin Flow cytometry Type of Flow cytometry Cytoflex S Hardware adaptation to ~100nm EV's The optical setup of the flow cytometer was modified to use the side scatter (SSC) information of the 405 nm laser (violet-SSC-A) instead of the 488 nm laser. The SSC was then calibrated using polystyrene beads of known diameter between 80 and 300 nm with a density of 1,05 g/cm_ and a refractive index of 1.59 nm (Cat 30080A, 30100A, 30200A and 30300A, Nanosphere serie 3000/ Thermofisher Scientific, Waltham, Massachusetts, USA). The forward scatter (FSC) and violet SSC-A were corrected on a logarithmic scale and the fluorescence channels were corrected on a logarithmic gain. The EV detection region was then set for events with size (by FSC) and complexity (by violet-SSC-A) characteristics of EVs. The SSC data generated by beads were fitted to nm according to Mie theory, using FCMPASS software (https://nano.ccr.cancer.gov/fcmpass/). Commercially available recombinant exosomes expressing green fluorescent protein (GFP) on their membrane surface (SAE0193, Merck) with a size distribution ranging from 30 to 200 nm (peak at 100-150 nm, measured by DLS) were used to validate the accuracy of the flow cytometer for the analysis of sEVs. Calibration bead size 0.08/ 0.1/ 0.2/ 0.3 Detected EV-associated proteins CD9/ CD63/ CD81/ HSP90/ CD44 Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 142 EM EM-type Transmission-EM Image type Wide-field

	EV230572	2/6	Homo sapiens	HEK293	PEI precipitation	Djeungoue-Petga, Marie-Ange	2024	50%
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 50% (87th 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 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 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 Not detected EV-associated proteins 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 Not detected EV-associated proteins GFP Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240006	1/4	Mus musculus	Neuro-2a (N2a) CCL-131	(d)(U)C Total Exosome Isolation lipid-based affinity capture	Weerakkody, Jonathan S.	2024	44%
Study summary Full title Photosensitive Nanoprobes for Rapid High Purity Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations All authors Jonathan S. Weerakkody, Tiffany Tseng, Mackenzie Topper, Sikha Thoduvayil, Abhijith Radhakrishnan, Frederic Pincet, Themis R. Kyriakides, Roshan W. Gunasekara, Sathish Ramakrishnan Journal Abstract The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as (show more...)The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. (hide) EV-METRIC 44% (84th 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 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 Total Exosome Isolation lipid-based affinity capture Protein markers EV: CD9/ CD63/ CD81/ Syt1 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Neuro-2a (N2a) CCL-131 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 12500 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Other Name other separation method lipid-based affinity capture EV-subtype Distinction between multiple subtypes Size Used subtypes 30-150 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ Syt1 Detected EV-associated proteins CD9/ CD63/ Syt1 Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 100-150 Extra information This paper was to validate the efficacy of photosensitive lipid nanoprobe for the isolation and size selective enrichment of native extracellular vesicles

	EV240006	2/4	Mus musculus	Primary bone marrow-derived macrophage (BMDM) cells from hind limbs in C57BL/6J wild type mice	(d)(U)C Total Exosome Isolation lipid-based affinity capture	Weerakkody, Jonathan S.	2024	44%
Study summary Full title Photosensitive Nanoprobes for Rapid High Purity Isolation and Size-Specific Enrichment of Synthetic and Extracellular Vesicle Subpopulations All authors Jonathan S. Weerakkody, Tiffany Tseng, Mackenzie Topper, Sikha Thoduvayil, Abhijith Radhakrishnan, Frederic Pincet, Themis R. Kyriakides, Roshan W. Gunasekara, Sathish Ramakrishnan Journal Abstract The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as (show more...)The biggest challenge in current isolation methods for lipid bilayer-encapsulated vesicles, such as exosomes, secretory, and synthetic vesicles, lies in the absence of a unified approach that seamlessly delivers high purity, yield, and scalability for large-scale applications. To address this gap, an innovative method is developed that utilizes photosensitive lipid nanoprobes for the efficient isolation of vesicles and sorting them into subpopulations based on size. The photosensitive component in the probe undergoes cleavage upon exposure to light, facilitating the release of vesicles in their near-native form. The method demonstrates a superior ability in isolating high purity extracellular vesicles from complex biological media and separating them into size-based subpopulations within 1 h, achieving more efficiency and purity than ultracentrifugation. Furthermore, this method's cost-effectiveness and rapid enrichment of the vesicles align with demands for large-scale isolation and downstream analyses of nucleic acids and proteins. The method opens new avenues in exploring, analyzing, and utilizing synthetic and extracellular vesicle subpopulations in various biomedical applications, including diagnostics, therapeutic delivery, and biomarker discovery. (hide) EV-METRIC 44% (84th 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 Total Exosome Isolation lipid-based affinity capture Protein markers EV: CD9/ CD63/ CD81/ Syt1 non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Primary bone marrow-derived macrophage (BMDM) cells from hind limbs in C57BL/6J wild type mice EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 12500 Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Other Name other separation method lipid-based affinity capture EV-subtype Distinction between multiple subtypes Size Used subtypes 30-200 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63/ CD81/ Syt1 Detected EV-associated proteins CD9/ CD63/ Syt1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 220-350 Extra information This paper was to validate the efficacy of photosensitive lipid nanoprobe for the isolation and size selective enrichment of native extracellular vesicles

	EV230996	2/5	Homo sapiens	HEK293F	(d)(U)C Filtration	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 Filtration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield per milliliter of starting sample

	EV230996	3/5	Homo sapiens	HEK293F	(d)(U)C Filtration UF	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 Filtration Ultrafiltration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield per milliliter of starting sample

	EV230996	4/5	Homo sapiens	HEK293F	(d)(U)C DC Filtration UF	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 cushion Filtration Ultrafiltration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Density cushion Density medium Sucrose Sample volume 20 Cushion volume 5 Density of the cushion 30% Centrifugation time 240 Centrifugation speed 120,000 Size-exclusion chromatography Resin type Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA Particle yield per milliliter of starting sample

	EV230996	5/5	Homo sapiens	HEK293F	(d)(U)C DC Filtration UF	Vo N	2024	44%
Study summary Full title A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles. All authors Vo N, Tran C, Tran NHB, Nguyen NT, Nguyen T, Ho DTK, Nguyen DDN, Pham T, Nguyen TA, Phan HTN, Nguyen HN, Tu LN Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompat (show more...)Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications. (hide) EV-METRIC 44% (84th 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 cushion Filtration Ultrafiltration Protein markers EV: CD9/ CD81/ TSG101/ CD63 non-EV: CANX Proteomics no Show all info Study aim New methodological development/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293F EV-harvesting Medium Serum free medium Cell count 3.00E+06 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 50.2 Ti Pelleting: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Density cushion Density of the cushion 30% Centrifugation speed 120,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected contaminants CANX Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Not detected contaminants CANX Characterization: Lipid analysis No Characterization: Particle analysis NTA

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