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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230058	1/2	Homo sapiens	NK92-MI	(d)(U)C Filtration UF SEC (non-commercial)	St-Denis-Bissonnette F	2023	63%
Study summary Full title A clinically relevant large-scale biomanufacturing workflow to produce natural killer cells and natural killer cell-derived extracellular vesicles for cancer immunotherapy. All authors St-Denis-Bissonnette F, Cummings SE, Qiu S, Stalker A, Muradia G, Mehic J, Mediratta K, Kaczmarek S, Burger D, Lee SH, Wang L, Lavoie JR Journal J Extracell Vesicles Abstract Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biothe (show more...)Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune-modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical-grade NK-EVs for pre-clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed-loop hollow-fibre bioreactor to continuously produce NK-EVs from the clinically relevant NK92-MI cell line under serum-free, Xeno-free and feeder-free conditions following GMP-compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK-EV biotherapeutics at the end of each production lot (over 10 viable cells and 10 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro-inflammatory cytokine (interferon-gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off-target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell-based and NK-EV-based immunotherapies for cancer with GMP considerations. (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 Filtration Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: Flotillin-1/ GAPDH/ GZMB non-EV: GM130/ Calnexin Proteomics no Show all info Study aim Function/Biomarker/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells NK92-MI EV-harvesting Medium Serum free medium Cell viability (%) 78 Cell count 9.26E+08 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 Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 9.4 Sample volume/column (mL) 51 Resin type None of these Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins Flotillin-1/ GAPDH/ GZMB Not detected contaminants GM130/ Calnexin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins MACSPlex Exosome kit human/ MACSPlex Cytokine Cytotoxic T/NK Cell Kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 72.80-164.00 EV concentration Yes Particle yield particles per milliliter of starting sample: 4.62E+11 EM EM-type Transmission-EM Image type Close-up Extra information A clinically relevant large-scale biomanufacturing workflow to produce Natural Killer cells and Natural Killer cell-derived extracellular vesicles for cancer immunotherapy

	EV230010	1/1	Homo sapiens	adipose-derived stem cells	UF qEV	Symonds, Emma	2023	63%
Study summary Full title Adipose derived stem cell extracellular vesicles modulate primary human macrophages to an anti-inflammatory phenotype in vitro All authors Emma K. C. Symonds, Bianca Black, Alexander Brown, Ineke Meredith, Margaret J. Currie, Kathryn E. Hally, Kirsty M. Danielson Journal J Extracell Biol Abstract EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue re (show more...)EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair because of their purported immune-regulatory properties. Extracellular vesicles (EVs) from ADSCs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are variable. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro. ADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from culture media and characterised by tuneable resistive pulse sensing, transmission electron microscopy and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFNγ), M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and ADSC-EVs were co-cultured with macrophages for 48 h. Flow cytometry and high-dimensional analysis clustered macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. Following the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster with a ‘high inflammatory profile’ (CD36+++CD206+++CD86+++; 16.5 ± 7.0%; p < 0.0001) to a cluster with a ‘lower inflammatory profile’ (CD36+CD206+CD86+; 35 ± 21.5%; p < 0.05). M1-like macrophages shifted from a cluster with a ‘high inflammatory profile’ (CD206++CD11b++CD36++CD163++; 26.1 ± 9.4%; p = 0.0024) to a ‘lower inflammatory profile’ (CD206+CD11b+CD36+CD163+; 72.8 ± 8.7%; p = 0.0007). There was no shift in M2-like clusters following ADSC-EV treatment. ADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state. (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 qEV Protein markers EV: CD9/ TSG101 non-EV: Apolipoprotein B/ Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adipose-derived stem cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 100 Cell count 1000000 Separation Method Ultra filtration Cut-off size (kDa) 100 Membrane type Cellulose acetate Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ TSG101 Not detected contaminants Apolipoprotein B/ Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 118 EV concentration Yes Particle yield particles per milliliter of starting sample: 30000000000 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220323	2/2	Homo sapiens	Serum	exoEasy	Kim JA	2023	63%
Study summary Full title Small RNA sequencing of circulating small extracellular vesicles microRNAs in patients with amyotrophic lateral sclerosis. All authors Kim JA, Park C, Sung JJ, Seo DJ, Choi SJ, Hong YH Journal Sci Rep Abstract Dysregulation of microRNAs (miRNA) in small extracellular vesicles (sEV) such as exosomes have been (show more...)Dysregulation of microRNAs (miRNA) in small extracellular vesicles (sEV) such as exosomes have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Although circulating cell-free miRNA have been extensively investigated in ALS, sEV-derived miRNAs have not been systemically explored yet. Here, we performed small RNA sequencing analysis of serum sEV and identified 5 differentially expressed miRNA in a discovery cohort of 12 patients and 11 age- and sex-matched healthy controls (fold change > 2, p < 0.05). Two of them (up- and down-regulation of miR-23c and miR192-5p, respectively) were confirmed in a separate validation cohort (18 patients and 15 healthy controls) by droplet digital PCR. Bioinformatic analysis revealed that these two miRNAs interact with distinct sets of target genes and involve biological processes relevant to the pathomechanism of ALS. Our results suggest that circulating sEV from ALS patients have distinct miRNA profiles which may be potentially useful as a biomarker of the disease. (hide) EV-METRIC 63% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Amyotrophic lateral sclerosis 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 exoEasy Protein markers EV: CD63/ CD81 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit exoEasy Other Name other separation method exoEasy Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type RNA-sequencing/ droplet digital PCR Database Vesiclepedia Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 138 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 138

	EV220321	1/8	Homo sapiens	SKMEL-37	(d)(U)C DG UF SEC (non-commercial)	Kashkanova AD	2023	63%
Study summary Full title Label-free discrimination of extracellular vesicles from large lipoproteins. All authors Kashkanova AD, Blessing M, Reischke M, Baur JO, Baur AS, Sandoghdar V, Van Deun J Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accur (show more...)Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accurate sizing and quantification of EVs remain problematic, given their nanometre size range and small scattering cross-sections. This is compounded by the fact that common EV isolation methods result in co-isolation of particles with comparable features. Especially in blood plasma, similarly-sized lipoproteins outnumber EVs to a great extent. Recently, interferometric nanoparticle tracking analysis (iNTA) was introduced as a particle analysis method that enables determining the size and refractive index of nanoparticles with high sensitivity and precision. In this work, we apply iNTA to differentiate between EVs and lipoproteins, and compare its performance to conventional nanoparticle tracking analysis (NTA). We show that iNTA can accurately quantify EVs in artificial EV-lipoprotein mixtures and in plasma-derived EV samples of varying complexity. Conventional NTA could not report on EV numbers, as it was not able to distinguish EVs from lipoproteins. iNTA has the potential to become a new standard for label-free EV characterization in suspension. (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 Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.1-1.2 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SKMEL-37 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >=100,000g Cell count 2.00E+08 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 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) 11.5 Sample volume (mL) 0.5 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 ELISA Antibody details provided? No Detected EV-associated proteins CD63 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 126 EV concentration Yes Other particle analysis name(1) interferometric nanoparticle tracking analysis Report type Median Report size 97 EV-concentration Yes Particle yield No

	EV230596	1/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Brain-EVP-1 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Albumin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Calreticulin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Calreticulin Not detected contaminants Albumin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Detected contaminants Calreticulin Not detected contaminants Albumin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 8.46E+11 Extra information Survey of organ-specific small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum

	EV230596	2/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Brain-EVP-2 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Albumin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Calreticulin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Calreticulin Not detected contaminants Albumin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Detected contaminants Calreticulin Not detected contaminants Albumin/ Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 3.02E+11

	EV230596	3/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Liver-EVP-1 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Calreticulin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 3.78E+12

	EV230596	4/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Liver-EVP-2 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Calreticulin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.19E+10

	EV230596	5/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Lung-EVP-1 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Calreticulin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.42E+12 Particle/mL

	EV230596	6/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Lung-EVP-2 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Albumin/ Calreticulin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.59E+12

	EV230596	7/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Herat-EVP-1 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Prohibitin Not detected contaminants Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.45E+11

	EV230596	8/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Herat-EVP-2 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Calreticulin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 8.22E+10

	EV230596	9/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Kidney-EVP-1 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Prohibitin Not detected contaminants Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 7.55E+11

	EV230596	10/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Kidney-EVP-2 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Calreticulin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 7.09E+11

	EV230596	11/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Fat-EVP-1 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ Calreticulin/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.26E+11

	EV230596	12/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Fat-EVP-2 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Calreticulin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.77E+11

	EV230596	13/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Serum-EVP-1 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein/ Albumin/ Calreticulin/ Prohibitin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.19E+10 Particle/mL

	EV230596	14/14	Mus musculus	Tissue	(d)(U)C	Abdelmohsen K	2023	57%
Study summary Full title Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum. All authors Abdelmohsen K, Herman AB, Carr AE, Henry-Smith CA, Rossi M, Meng Q, Yang JH, Tsitsipatis D, Bangura A, Munk R, Martindale JL, Nogueras-Ortiz CJ, Hao J, Gong Y, Liu Y, Cui CY, Hartnell LM, Price NL, Ferrucci L, Kapogiannis D, de Cabo R, Gorospe M Journal J Extracell Biol Abstract Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different ci (show more...)Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age. (hide) EV-METRIC 57% (45th 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 Tissue Sample origin Serum-EVP-2 Focus vesicles extracellular vesicle and particles 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/ CD81 non-EV: Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein/ Albumin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 12 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin/ Calreticulin/ Prohibitin Not detected contaminants Argonaute-2/ GM130/ PMP70/ Tamm-Horsfall protein Detected EV-associated proteins CD9/ CD81 Not detected contaminants Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report size (nm) 50-100 Other particle analysis name(3) Nanoflow cytometry Report type Size range/distribution Report size 50-100 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.19E+10 Partic/mL

	EV220407	1/1	Homo sapiens	adult primary astrocytes	(d)(U)C	Shanthi KB	2023	57%
Study summary Full title Human Adult Astrocyte Extracellular Vesicle Transcriptomics Study Identifies Specific RNAs Which Are Preferentially Secreted as EV Luminal Cargo. All authors Shanthi KB, Fischer D, Sharma A, Kiviniemi A, Kaakinen M, Vainio SJ, Bart G Journal Genes (Basel) Abstract Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and (show more...)Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs. (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 (Differential) (ultra)centrifugation Protein markers EV: CD9/ CD63/ CD81/ Syntenin non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells adult primary astrocytes EV-harvesting Medium Serum free medium 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 AH-629 (36 ml) Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Fluorometric assay Protein Yield (µg) per milliliter of starting sample Other 2 Exoview Detected EV-associated proteins CD9/ CD63/ CD81/ Syntenin Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 660 µg/ml RNAse treatment Yes RNAse type RNase A and RNase T1 RNAse concentration 0.5 mg/ml Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 107.9 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.20E+06 EM EM-type Transmission-EM/ Immuno-EM EM protein Integrin beta1 Image type Close-up, Wide-field Report size (nm) 50 to 200 nm EV concentration Non

	EV220311	1/1	Homo sapiens	erythrocyte	(d)(U)C SEC (non-commercial)	Singh, Priyanka	2023	57%
Study summary Full title Removal and identification of external protein corona members from RBC-derived extracellular vesicles by surface manipulating antimicrobial peptides All authors Priyanka Singh, Imola Cs. Szigyártó, Maria Ricci, Anikó Gaál, Mayra Maritza Quemé-Peña, Diána Kitka, Lívia Fülöp, Lilla Turiák, László Drahos, Zoltán Varga, Tamás Beke-Somfai Journal Journal of Extracellular Biology Abstract In the last years, extracellular vesicles (EVs), secreted by various cells and body fluids have show (show more...)In the last years, extracellular vesicles (EVs), secreted by various cells and body fluids have shown extreme potential in biomedical applications. Increasing number of studies suggest that a protein corona could adhere to the surface of EVs which can have a fundamental effect on their function, targeting and therapeutical efficacy. However, removing and identifying these corona members is currently a challenging task to achieve. In this study we have employed red blood cell-derived extracellular vesicles (REVs) as a model system and three membrane active antimicrobial peptides (AMPs), LL-37, FK-16 and CM15, to test whether they can be used to remove protein corona members from the surface of vesicles. These AMPs were reported to preferentially exert their membrane-related activity via one of the common helical surface-covering models and do not significantly affect the interior of lipid bilayer bodies. The interaction between the peptides and the REVs was followed by biophysical techniques, such as flow-linear dichroism spectroscopy which provided the effective applicable peptide concentration for protein removal. REV samples were then subjected to subsequent size exclusion chromatography and to proteomics analysis. Based on the comparison of control REVs with the peptide treated samples, seventeen proteins were identified as external protein corona members. From the three investigated AMPs, FK-16 can be considered as the best candidate to further optimize EV-related applicability of AMPs. Our results on the REV model system envisage that membrane active peptides may become a useful set of tools in engineering and modifying surfaces of EVs and other lipid-based natural particles. (hide) EV-METRIC 57% (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 erythrocyte 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 Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: Albumin Proteomics yes 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 erythrocyte Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Eppendord F45-21-11 Pelleting: speed (g) 16000 Size-exclusion chromatography Used for validation? Yes Total column volume (mL) 3.5 Sample volume/column (mL) 0.1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per milliliter of starting sample Proteomics database No Detected contaminants Albumin Characterization: Lipid analysis No Characterization: Particle analysis DLS EM EM-type Transmission-EM Image type Close-up Other particle analysis name(1) Microfluidic resistive pulse sensing Report type Mean Report size 162 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.30E+12

	EV230993	1/2	Homo sapiens	bone marrow derived mesenchymal stromal cells	(d)(U)C	Eslami N	2023	56%
Study summary Full title Regenerative potential of different extracellular vesicle subpopulations derived from clonal mesenchymal stem cells in a mouse model of chemotherapy-induced premature ovarian failure. All authors Eslami N, Bahrehbar K, Esfandiari F, Shekari F, Hassani SN, Nazari A, Pakzad M, Baharvand H Journal Life Sci Abstract Some studies have shown that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (show more...)Some studies have shown that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (MSC-EVs) can restore ovarian function in premature ovarian failure (POF), however, concerns about their efficacy are attributed to the heterogeneity of the cell populations and EVs. Here, we assessed the therapeutic potential of a homogeneous population of clonal MSCs (cMSCs) and their EVs subpopulations in a mouse model of POF. (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: CD63/ CD81/ TSG101 non-EV: Calnexin Proteomics no Show all info Study aim Function/New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bone marrow derived mesenchymal stromal cells EV-harvesting Medium Serum-containing medium Cell viability (%) 95 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) 110000 Wash: volume per pellet (ml) 30 Wash: time (min) 120 Wash: Rotor Type SW 32 Ti Wash: speed (g) 110000 Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 186

	EV230981	1/6	Mus musculus	Blood plasma	(d)(U)C	André-Grégoire G	2023	56%
Study summary Full title Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation. All authors André-Grégoire G, Roux Q, Gavard J Journal STAR Protoc Abstract Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological co (show more...)Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).. (hide) EV-METRIC 56% (88th 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: Alix/ CD9/ HSP70 non-EV: Calreticulin/ GM130/ Albumin Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 2 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ HSP70 Detected contaminants Albumin Not detected contaminants Calreticulin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230981	4/6	Mus musculus	Blood plasma	(d)(U)C	André-Grégoire G	2023	56%
Study summary Full title Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation. All authors André-Grégoire G, Roux Q, Gavard J Journal STAR Protoc Abstract Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological co (show more...)Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).. (hide) EV-METRIC 56% (88th 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 bearing human GSC-derived orthotopic tumour 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/ HSP70 non-EV: ApoB Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 2 Wash: time (min) 120 Wash: Rotor Type SW 41 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ HSP70 Detected contaminants ApoB Characterization: Lipid analysis No Characterization: Particle analysis Other particle analysis name(1) Interferometric light microscopy Report type Median Report size 197 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 5.36E9

	EV230973	1/4	Homo sapiens	DKs-8	(d)(U)C DC DG	Jimenez L	2023	56%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (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 Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DKs-8 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.13E Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield total particles in 50 microliter: 1.24E+11

	EV230973	4/4	Homo sapiens	DLD-1	(d)(U)C DC DG	Jimenez L	2023	56%
Study summary Full title Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations. All authors Jimenez L, Barman B, Jung YJ, Cocozza L, Krystofiak E, Saffold C, Vickers KC, Wilson JT, Dawson TR, Weaver AM Journal J Extracell Vesicles Abstract Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in (show more...)Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination. (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 Density cushion Density gradient Adj. k-factor 3.13 (washing) Protein markers EV: CD63/ Flotillin-1 non-EV: None Proteomics no EV density (g/ml) 1.14 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Wash: volume per pellet (ml) 3 Wash: time (min) 30 Wash: Rotor Type TLA-110 Wash: speed (g) 10000 Wash: adjusted k-factor 3.13E Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 3.13E Density cushion Density medium Iodixanol Sample volume 30 Cushion volume 2 Density of the cushion 60% Centrifugation time 240 Centrifugation speed 100000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 145 EV concentration Yes Particle yield total particles in 50 microliter: 21200000000

	EV230968	1/2	Homo sapiens	umbilical cord mesenchymal stem cells	(d)(U)C	Xie W	2023	56%
Study summary Full title TNF-α preconditioned UCMSCs-derived EVs enhance the inhibition of necroptosis of acinar cells in SAP. All authors Xie W, Luo T, Ma Z, Xue S, Jia X, Yang T, Song Z Journal Tissue Eng Part A Abstract Severe acute pancreatitis (SAP) is a common abdominal emergency with a high mortality rate and a lac (show more...)Severe acute pancreatitis (SAP) is a common abdominal emergency with a high mortality rate and a lack of effective therapeutic options. Although mesenchymal stem cells (MSCs) transplantation is a potential treatment for SAP, the mechanism remains unclear. It has been suggested that MSCs may act mainly through paracrine effects/ therefore, we aimed to demonstrate the therapeutic efficacy of extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (UCMSCs) for SAP. Na-taurocholate (NaT) was used to induce a rat SAP model via retrograde injection into the common biliopancreatic duct. After 72 hours of EVs transplantation, pancreatic pathological damage was alleviated, along with a decrease in serum amylase activity and pro-inflammatory cytokines levels. Interestingly, when UCMSCs were preconditioned with 10ng/mL TNF-α for 48h, the obtained EVs (named TNF-α-EVs) performed an enhanced efficacy. Furthermore, both animal and cellular experiments showed that TNF-α-EVs alleviated the necroptosis of acinar cells of SAP via RIPK3/MLKL axis. In conclusion, our study demonstrated that TNF-α-EVs was able to enhance the therapeutic effect on SAP by inhibiting necroptosis compared to normal EVs. This study heralds that TNF-α-EVs may be a promising therapeutic approach for SAP in the future. (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/ CD63/ TSG101/ Syntenin non-EV: Calnexin Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells umbilical cord mesenchymal stem cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 6000000 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 25 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ TSG101/ Syntenin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 149.6 EM EM-type Transmission-EM Image type Wide-field

	EV230968	2/2	Homo sapiens	umbilical cord mesenchymal stem cells	(d)(U)C	Xie W	2023	56%
Study summary Full title TNF-α preconditioned UCMSCs-derived EVs enhance the inhibition of necroptosis of acinar cells in SAP. All authors Xie W, Luo T, Ma Z, Xue S, Jia X, Yang T, Song Z Journal Tissue Eng Part A Abstract Severe acute pancreatitis (SAP) is a common abdominal emergency with a high mortality rate and a lac (show more...)Severe acute pancreatitis (SAP) is a common abdominal emergency with a high mortality rate and a lack of effective therapeutic options. Although mesenchymal stem cells (MSCs) transplantation is a potential treatment for SAP, the mechanism remains unclear. It has been suggested that MSCs may act mainly through paracrine effects/ therefore, we aimed to demonstrate the therapeutic efficacy of extracellular vesicles (EVs) derived from human umbilical cord mesenchymal stem cells (UCMSCs) for SAP. Na-taurocholate (NaT) was used to induce a rat SAP model via retrograde injection into the common biliopancreatic duct. After 72 hours of EVs transplantation, pancreatic pathological damage was alleviated, along with a decrease in serum amylase activity and pro-inflammatory cytokines levels. Interestingly, when UCMSCs were preconditioned with 10ng/mL TNF-α for 48h, the obtained EVs (named TNF-α-EVs) performed an enhanced efficacy. Furthermore, both animal and cellular experiments showed that TNF-α-EVs alleviated the necroptosis of acinar cells of SAP via RIPK3/MLKL axis. In conclusion, our study demonstrated that TNF-α-EVs was able to enhance the therapeutic effect on SAP by inhibiting necroptosis compared to normal EVs. This study heralds that TNF-α-EVs may be a promising therapeutic approach for SAP in the future. (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 TNFa treatment (10ng/ml) 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/ CD63/ TSG101/ Syntenin non-EV: Calnexin Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells umbilical cord mesenchymal stem cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 6000000 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 Type 70 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 25 Wash: time (min) 90 Wash: Rotor Type Type 70 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ TSG101/ Syntenin Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 150.5 EM EM-type Transmission-EM Image type Wide-field

	EV230567	1/2	Homo sapiens	Blood plasma	(d)(U)C	Li L	2023	56%
Study summary Full title Circulating extracellular vesicles from patients with traumatic brain injury induce cerebrovascular endothelial dysfunction. All authors Li L, Li F, Bai X, Jia H, Wang C, Li P, Zhang Q, Guan S, Peng R, Zhang S, Dong JF, Zhang J, Xu X Journal Pharmacol Res Abstract Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury ( (show more...)Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury (TBI). We previously demonstrated that extracellular vesicles (EVs) released from injured brains led to endothelial barrier disruption and vascular leakage. However, the molecular mechanisms of this EV-induced endothelial dysfunction (endotheliopathy) remain unclear. Here, we enriched plasma EVs from TBI patients (TEVs), and detected high mobility group box 1 (HMGB1) exposure to 50.33 ± 10.17% of TEVs and the number of HMGB1TEVs correlated with injury severity. We then investigated for the first time the impact of TEVs on endothelial function using adoptive transfer models. We found that TEVs induced dysfunction of cultured human umbilical vein endothelial cells and mediated endothelial dysfunction in both normal and TBI mice, which were propagated through the HMGB1-activated receptor for advanced glycation end products (RAGE)/Cathepsin B signaling, and the resultant NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and canonical caspase-1/gasdermin D (GSDMD)-dependent pyroptosis. Finally, von Willebrand factor (VWF) was detected on the surface of 77.01 ± 7.51% of HMGB1TEVs. The TEV-mediated endotheliopathy was reversed by a polyclonal VWF antibody, indicating that VWF might serve a coupling factor that tethered TEVs to ECs, thus facilitating HMGB1-induced endotheliopathy. These results suggest that circulating EVs isolated from patients with TBI alone are sufficient to induce endothelial dysfunction and contribute to secondary brain injury that are dependent on immunologically active HMGB1 exposed on their surface. This finding provided new insight for the development of potential therapeutic targets and diagnostic biomarkers for TBI. (hide) EV-METRIC 56% (88th 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: CD9/ CD81/ TSG101/ Calnexin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 0.1 Wash: time (min) 60 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected EV-associated proteins Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 119.2±10.09 Particle analysis: flow cytometry Flow cytometer type FACS LSR II flow cytometer Hardware adjustment - Calibration bead size 0.5/ 0.9/ 3 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.32±1.82E07 and 2.4 ± 0.69E07 EM EM-type Transmission-EM Image type Close-up

	EV230567	2/2	Homo sapiens	Blood plasma	(d)(U)C	Li L	2023	56%
Study summary Full title Circulating extracellular vesicles from patients with traumatic brain injury induce cerebrovascular endothelial dysfunction. All authors Li L, Li F, Bai X, Jia H, Wang C, Li P, Zhang Q, Guan S, Peng R, Zhang S, Dong JF, Zhang J, Xu X Journal Pharmacol Res Abstract Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury ( (show more...)Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury (TBI). We previously demonstrated that extracellular vesicles (EVs) released from injured brains led to endothelial barrier disruption and vascular leakage. However, the molecular mechanisms of this EV-induced endothelial dysfunction (endotheliopathy) remain unclear. Here, we enriched plasma EVs from TBI patients (TEVs), and detected high mobility group box 1 (HMGB1) exposure to 50.33 ± 10.17% of TEVs and the number of HMGB1TEVs correlated with injury severity. We then investigated for the first time the impact of TEVs on endothelial function using adoptive transfer models. We found that TEVs induced dysfunction of cultured human umbilical vein endothelial cells and mediated endothelial dysfunction in both normal and TBI mice, which were propagated through the HMGB1-activated receptor for advanced glycation end products (RAGE)/Cathepsin B signaling, and the resultant NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and canonical caspase-1/gasdermin D (GSDMD)-dependent pyroptosis. Finally, von Willebrand factor (VWF) was detected on the surface of 77.01 ± 7.51% of HMGB1TEVs. The TEV-mediated endotheliopathy was reversed by a polyclonal VWF antibody, indicating that VWF might serve a coupling factor that tethered TEVs to ECs, thus facilitating HMGB1-induced endotheliopathy. These results suggest that circulating EVs isolated from patients with TBI alone are sufficient to induce endothelial dysfunction and contribute to secondary brain injury that are dependent on immunologically active HMGB1 exposed on their surface. This finding provided new insight for the development of potential therapeutic targets and diagnostic biomarkers for TBI. (hide) EV-METRIC 56% (88th 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 Traumatic brain injury 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/ CD81/ TSG101/ Calnexin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 0.1 Wash: time (min) 60 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD81/ TSG101 Not detected EV-associated proteins Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 118.4±6.98 Particle analysis: flow cytometry Flow cytometer type FACS LSR II flow cytometer Hardware adjustment nanoscale flow cytometry Calibration bead size 0.5/ 0.9/ 3 EV concentration Yes Particle yield particles per milliliter of starting sample: 2.4±0.69E07 EM EM-type Transmission-EM Image type Close-up

	EV230374	1/13	Homo sapiens	HEK293T	(d)(U)C	Levy-Myers R	2023	56%
Study summary Full title An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function. All authors Levy-Myers R, Daudelin D, Na CH, Sockanathan S Journal Sci Adv Abstract Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the (show more...)Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [ knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in KOs, while EVs from KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape. (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: ANXA1/ CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium 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) 12000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins ANXA1 Not detected EV-associated proteins CD63/ CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 225 EV concentration Yes Particle yield Not reported EM EM-type Transmission-EM Image type Wide-field

	EV230374	3/13	Homo sapiens	HEK293T	(d)(U)C	Levy-Myers R	2023	56%
Study summary Full title An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function. All authors Levy-Myers R, Daudelin D, Na CH, Sockanathan S Journal Sci Adv Abstract Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the (show more...)Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [ knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in KOs, while EVs from KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape. (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 GDE3 overexpression Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Calnexin/ GDE3/ CD63/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Function/Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293T EV-harvesting Medium Serum free medium 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) 12000 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ ANXA1/ Actin/ GDE3 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield Not reported EM EM-type Transmission-EM Image type Wide-field

	EV230059	18/25	Homo sapiens	Blood plasma	(d)(U)C	Irmer B	2023	56%
Study summary Full title Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression. All authors Irmer B, Efing J, Reitnauer LE, Angenendt A, Heinrichs S, Schubert A, Schulz M, Binder C, Tio J, Hansen U, Geyer C, Gerwing M, Bleckmann A, Menck K Journal Cell Commun Signal Abstract Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport a (show more...)Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. (hide) EV-METRIC 56% (88th 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 breast cancer 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 Protein markers EV: actin-beta/ RGAP1/ Actinin-4/ ROR2/ EpCAM/ ROR1 non-EV: ApoB/ ApoA1 Proteomics no Show all info Study aim Function/Biomarker/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation 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 Filtration steps Below or equal to 800/ Between 800 and 10,000/ Equal to or above 10,000 and below 50,000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins actin-beta/ RGAP1/ Actinin-4 Detected contaminants ApoA1 Not detected contaminants ApoB Flow cytometry Type of Flow cytometry Standard flow cytometer Calibration bead size 0.8/ 0.5 Antibody details provided? No Detected EV-associated proteins ROR2/ EpCAM/ ROR1 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 214.9

	EV230053	1/2	Homo sapiens	Blood plasma	(d)(U)C	Bettio V	2023	56%
Study summary Full title Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques. All authors Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D Journal PLoS One Abstract Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide) EV-METRIC 56% (88th 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: CD9/ CD63/ CD81/ HSP70/ MHC1 non-EV: Histones/ Albumin/ APOB48/B100/ APOA1 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 Blood plasma 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) 146 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Detected contaminants Albumin/ APOB48/B100/ APOA1 Not detected contaminants Histones Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ MHC1 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 100-170 EV concentration Yes Particle yield particles per milliliter of starting sample: 1000000-25000000

	EV230053	2/2	Homo sapiens	Blood plasma	(d)(U)C	Bettio V	2023	56%
Study summary Full title Extracellular vesicles from human plasma for biomarkers discovery: Impact of anticoagulants and isolation techniques. All authors Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D Journal PLoS One Abstract Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide) EV-METRIC 56% (88th 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: CD9/ CD63/ CD81/ HSP70/ MHC1 non-EV: Histones/ Albumin/ APOB48/B100/ APOA1 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 Blood plasma 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) 146 Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ HSP70 Detected contaminants Albumin/ APOB48/B100/ APOA1 Not detected contaminants Histones Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ MHC1 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 150-210 EV concentration Yes Particle yield particles per milliliter of starting sample: 12000000-68000000

	EV230008	6/42	Mus musculus	EO771	(d)(U)C DG UF	Cocozza F	2023	56%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (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 10k light 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 Ultrafiltration Protein markers EV: CD9/ CD63 non-EV: None Proteomics no EV density (g/ml) 1.015-1.085 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 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 MLA-80 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 22% Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 1 Orientation Top-down Speed (g) 187000 Duration (min) 90 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 5.73E Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Distinction between multiple subtypes Density Used subtypes 1.015-1.035 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230008	7/42	Mus musculus	EO771	(d)(U)C DG UF	Cocozza F	2023	56%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (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 10k dense 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 Ultrafiltration Protein markers EV: CD9/ CD63 non-EV: None Proteomics no EV density (g/ml) 1.015-1.085 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 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 MLA-80 Pelleting: speed (g) 10000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 22% Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 1 Orientation Top-down Speed (g) 187000 Duration (min) 90 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 5.73E Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Distinction between multiple subtypes Density Used subtypes 1.065-1.085 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230008	41/42	Mus musculus	myr/palm-OVA-EO771	(d)(U)C DG UF	Cocozza F	2023	56%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (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 myr/palm-OVA Focus vesicles sEV 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 Ultrafiltration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.015-1.085 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells myr/palm-OVA-EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type MLA-80 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 22% Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 1 Orientation Top-down Speed (g) 187000 Duration (min) 90 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 2.29E Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Distinction between multiple subtypes Density Used subtypes 1.015-1.035 Characterization: Protein analysis Protein Concentration Method microBCA Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230008	42/42	Mus musculus	myr/palm-OVA-EO771	(d)(U)C DG UF	Cocozza F	2023	56%
Study summary Full title Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells. All authors Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C Journal EMBO J Abstract Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (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 myr/palm-OVA Focus vesicles VLP 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 Ultrafiltration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.015-1.085 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells myr/palm-OVA-EO771 EV-harvesting Medium Serum free medium Cell viability (%) 85 Cell count 100000000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type MLA-80 Pelleting: speed (g) 200000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 6% Highest density fraction 22% Total gradient volume, incl. sample (mL) 16 Sample volume (mL) 1 Orientation Top-down Speed (g) 187000 Duration (min) 90 Fraction volume (mL) 2 Fraction processing Centrifugation Pelleting: volume per fraction 6 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 2.29E Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose EV-subtype Distinction between multiple subtypes Density Used subtypes 1.065-1.085 Characterization: Protein analysis Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220406	1/1	Equus caballus	Follicular fluid	(d)(U)C Filtration	Gebremedhn S	2023	56%
Study summary Full title Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation. All authors Gebremedhn S, Gad A, Ishak GM, Menjivar NG, Gastal MO, Feugang JM, Prochazka R, Tesfaye D, Gastal EL Journal Mol Hum Reprod Abstract Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerab (show more...)Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from equine FF aspirated in vivo during different stages of follicular development, namely, predeviation (PreDev/ 18-20 mm), deviation (Dev/ 22-25 mm), postdeviation (PostDev/ 26-29 mm), preovulatory (PreOV/ 30-35 mm), and impending ovulation (IMP/ ∼40 mm). Approximately 176 known miRNAs were found in all groups with 144 mutually detected among all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in expression from the PreDev until the PreOV stage. Another cluster of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression pattern at the PreDev stage until the PostDev stage, with a significant increase at the PreOV stage followed by a significant decrease at the IMP stage. In conclusion, this study provides greater insights into the stage-specific expression dynamics of FF EV-miRNAs during equine follicular development, which may propose novel approaches to improve ART and provide new biomarkers to facilitate the assessment of ovarian pathophysiological conditions. (hide) EV-METRIC 56% (83rd 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 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: CD63/ TSG101/ Flotillin-1 non-EV: CytC Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Equus caballus 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) 120,000 Wash: volume per pellet (ml) 2 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120,000 Filtration steps 0.2 or 0.22 ?m Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ Flotillin-1 Not detected contaminants CytC Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA EV concentration Yes Particle yield particles per milliliter of starting sample: 1.50E+08 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 91-111

	EV220402	1/1	Bos taurus	follicular fluid	(d)(U)C Filtration	Gad A	2023	56%
Study summary Full title Extracellular vesicle-microRNAs mediated response of bovine ovaries to seasonal environmental changes. All authors Gad A, Joyce K, Menjivar NG, Heredia D, Rojas CS, Tesfaye D, Gonella-Diaza A Journal J Ovarian Res Abstract Among the various seasonal environmental changes, elevated ambient temperature during the summer sea (show more...)Among the various seasonal environmental changes, elevated ambient temperature during the summer season is a main cause of stress in dairy and beef cows, leading to impaired reproductive function and fertility. Follicular fluid extracellular vesicles (FF-EVs) play an important role in intrafollicular cellular communication by, in part, mediating the deleterious effects of heat stress (HS). Here we aimed to investigate the changes in FF-EV miRNA cargoes in beef cows in response to seasonal changes: summer (SUM) compared to the winter (WIN) season using high throughput sequencing of FF-EV-coupled miRNAs. In addition to their biological relevance, the potential mechanisms involved in the packaging and release of those miRNAs as a response to environmental HS were elucidated. (hide) EV-METRIC 56% (83rd 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 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: CD63/ Flotillin-1/ TSG101 non-EV: CYCS Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Bos taurus 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) 4 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotillin-1/ TSG101 Not detected contaminants CYCS Characterization: RNA analysis RNA analysis Type RNA-sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 114 EV concentration Yes Particle yield particles per milliliter of starting sample: 3.5E11-1.75E12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220369	4/5	Homo sapiens	Blood plasma	(d)(U)C Filtration Total Exosome Isolation	Lapin M	2023	56%
Study summary Full title Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer. All authors Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O Journal PLoS One Abstract Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide) EV-METRIC 56% (88th 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 Filtration Commercial method Protein markers EV: CD9/ CD63/ CD81/ TSG101 non-EV: ApoA1 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods 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 No Filtration steps Larger than 0.45 µm Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81/ TSG101 Detected contaminants ApoA1 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 1-10000

	EV220318	3/4	Mus musculus	PK1	(d)(U)C	Jakub Soukup	2023	56%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small 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 Prion infected cell culture 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 Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ CD81/ PrP non-EV: Calnexin Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells PK1 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 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: rotor type SW 28 Pelleting: speed (g) 19827 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 19556.1 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ Integrin-beta1/ PrP Not detected EV-associated proteins CD81 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV220318	4/4	Mus musculus	PK1	(d)(U)C DC	Jakub Soukup	2023	56%
Study summary Full title Large extracellular vesicles transfer more prions and infect cell culture better than small extracellular vesicles All authors Jakub Soukup, Tibor Moško, Sami Kereïche, Karel Holada Journal Biochem Pharmacol Abstract Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans (show more...)Prions are responsible for a number of lethal neurodegenerative and transmissible diseases in humans and animals. Extracellular vesicles, especially small exosomes, have been extensively studied in connection with various diseases. In contrast, larger microvesicles are often overlooked. In this work, we compared the ability of large extracellular vesicles (lEVs) and small extracellular vesicles (sEVs) to spread prions in cell culture. We utilized CAD5 cell culture model of prion infection and isolated lEVs by 20,000×g force and sEVs by 110,000×g force. The lEV fraction was enriched in β-1 integrin with a vesicle size starting at 100 nm. The fraction of sEVs was partially depleted of β-1 integrin with a mean size of 79 nm. Both fractions were enriched in prion protein, but the lEVs contained a higher prion-converting activity. In addition, lEV infection led to stronger prion signals in both cell cultures, as detected by cell and western blotting. These results were verified on N2a-PK1 cell culture. Our data suggest the importance of lEVs in the trafficking and spread of prions over extensively studied small 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 Prion infected cell culture Focus vesicles small extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density cushion Protein markers EV: Alix/ CD9/ CD63/ HSP70/ TSG101/ CD81/ PrP non-EV: Calnexin/ Integrin-beta1 Proteomics no Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells PK1 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 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 28 Pelleting: speed (g) 112398.1 Wash: volume per pellet (ml) 13.5 Wash: time (min) 70 Wash: Rotor Type SW 40 Ti Wash: speed (g) 110862.3 Density cushion Density medium Sucrose Sample volume 11 Cushion volume 2.5 Density of the cushion 40% Centrifugation time 70 Centrifugation speed 110862.3 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63/ HSP70/ TSG101/ PrP Not detected EV-associated proteins CD81 Detected contaminants Integrin-beta1 Not detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field

	EV230005	1/4	Homo sapiens	Serum	(d)(U)C	Dobra G	2023	55%
Study summary Full title MMP-9 as Prognostic Marker for Brain Tumours: A Comparative Study on Serum-Derived Small Extracellular Vesicles. All authors Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K Journal Cancers (Basel) Abstract Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide) EV-METRIC 55% (91st 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 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/ CD5L/ MMP-9 non-EV: calnexin Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum 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 T-1270 Pelleting: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD5L Not detected contaminants calnexin ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins MMP-9 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 77.3 EV concentration Yes Particle yield particles per milliliter of starting sample: 1.44E+12 EM EM-type Transmission-EM Image type Close-up

	EV220319	3/6	Homo sapiens	Serum	(d)(U)C	Schmoldt A	2023	55%
Study summary Full title Small extracellular vesicles are released ex vivo from platelets into serum and from residual blood cells into stored plasma All authors Małgorzata S. Małys, Maximilian C. Köller, Kristin Papp, Christof Aigner, Daffodil Dioso, Patrick Mucher, Helga Schachner, Michael Bonelli, Helmuth Haslacher, Andrew J. Rees, Renate Kain Journal Journal of Extracellular Biology Abstract Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers (show more...)Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA-ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non-platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca2+, an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo. (hide) EV-METRIC 55% (91st 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 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 Protein markers EV: CD9/ CD63/ CD81 non-EV: Calnexin/ Albumin/ ApoB100 Proteomics no Show all info Study aim Biomarker/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Serum 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 P28S(SRP28SA) Swinging bucket rotor Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 32 Wash: time (min) 120 Wash: Rotor Type P28S(SRP28SA) Swinging bucket rotor Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Not detected EV-associated proteins CD9/ CD63 Detected contaminants Albumin Not detected contaminants Calnexin ELISA Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Detected contaminants ApoB100 Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ CD81 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) CD9/ CD63/ CD81/ MACSPlex exosome human kit Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 161.1 EV concentration Yes Particle yield per ml of purification EM EM-type Immuno-EM EM protein CD9 Image type Close-up

	EV230981	2/6	Mus musculus	Blood plasma	(d)(U)C qEVoriginal/70nm	André-Grégoire G	2023	50%
Study summary Full title Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation. All authors André-Grégoire G, Roux Q, Gavard J Journal STAR Protoc Abstract Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological co (show more...)Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).. (hide) EV-METRIC 50% (83rd 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 qEVoriginal/70nm Protein markers EV: Alix/ CD9/ HSP70 non-EV: Albumin/ Calreticulin/ GM130 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit qEVoriginal/70nm Other Name other separation method qEVoriginal/70nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9 Not detected EV-associated proteins HSP70 Not detected contaminants Albumin/ Calreticulin/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230981	5/6	Mus musculus	Blood plasma	(d)(U)C qEVoriginal/70nm	André-Grégoire G	2023	50%
Study summary Full title Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation. All authors André-Grégoire G, Roux Q, Gavard J Journal STAR Protoc Abstract Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological co (show more...)Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).. (hide) EV-METRIC 50% (83rd 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 bearing human GSC-derived orthotopic tumour 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 qEVoriginal/70nm Protein markers EV: Alix/ CD9/ HSP70 non-EV: ApoB Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Commercial kit qEVoriginal/70nm Other Name other separation method qEVoriginal/70nm Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ HSP70 Detected contaminants ApoB Characterization: Lipid analysis No Characterization: Particle analysis Other particle analysis name(1) Interferometric light microscopy Report type Median Report size 197 EV-concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.68E9

	EV230606	1/2	Homo sapiens	MDA-MB-231	Filtration qEV	Loconte L	2023	50%
Study summary Full title Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods. All authors Loconte L, Arguedas D, El R, Zhou A, Chipont A, Guyonnet L, Guerin C, Piovesana E, Vázquez-Ibar JL, Joliot A, Théry C, Martín-Jaular L Journal J Extracell Vesicles Abstract Cell-cell communication within the complex tumour microenvironment is critical to cancer progression (show more...)Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate. (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 Filtration qEV Protein markers EV: Alix/ CD9/ CD63 non-EV: Calreticulin/ 14-3-3 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 60000000 Separation Method Filtration steps 10 Other Name other separation method qEV Other Name other separation method Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63 Not detected contaminants Calreticulin/ 14-3-3 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield number of particles per million cells: E09 Particle analysis: flow cytometry Hardware adjustment Other particle analysis name(2) Nanoflow cytometry Report type Size range/distribution Report size 100

	EV230606	2/2	Homo sapiens	MDA-MB-231	Filtration qEV	Loconte L	2023	50%
Study summary Full title Detection of the interactions of tumour derived extracellular vesicles with immune cells is dependent on EV-labelling methods. All authors Loconte L, Arguedas D, El R, Zhou A, Chipont A, Guyonnet L, Guerin C, Piovesana E, Vázquez-Ibar JL, Joliot A, Théry C, Martín-Jaular L Journal J Extracell Vesicles Abstract Cell-cell communication within the complex tumour microenvironment is critical to cancer progression (show more...)Cell-cell communication within the complex tumour microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Deciphering the interactions between TD-EVs and immune cells is essential to understand immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection by imaging flow cytometry and multicolour spectral flow cytometry of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs). EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labelled either with the lipophilic dye MemGlow-488 (MG-488), Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) or through ectopic expression of a MyrPalm-superFolderGFP reporter (mp-sfGFP), which incorporates into EVs during their biogenesis. Our results show that these labelling strategies, although analysed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and mp-sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labelling method. Furthermore, our results show that the method used for EV labelling influences the detection of the different types of EV interactions with the recipient cells. Specifically, MG-488, CFSE and mp-sfGFP result in observation suggesting, respectively, transient EV-PM interaction that results in dye transfer, EV content delivery, and capture of intact EVs. Consequently, the type of EV labelling method has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate. (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 mp-sfGFP-MDA-MB-231 transduced cells 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 qEV Protein markers EV: Alix/ CD9/ CD63 non-EV: Calreticulin/ 14-3-3 Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDA-MB-231 EV-harvesting Medium Serum free medium Cell viability (%) 95 Cell count 60000000 Separation Method Filtration steps 10 Other Name other separation method qEV Other Name other separation method Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ CD63 Not detected contaminants Calreticulin/ 14-3-3 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield number of particles per million cells: E09 Particle analysis: flow cytometry Hardware adjustment Other particle analysis name(2) Nanoflow cytometry Report type Size range/distribution Report size 100

	EV230600	1/1	Homo sapiens	MCF7	Filtration UF	Pauwels, Jarne	2023	50%
Study summary Full title Filter-aided extracellular vesicle enrichment (FAEVEr) All authors Jarne Pauwels, Tessa Van de Steene, Jana Van de Velde, Sven Eyckerman, Kris Gevaert Journal Abstract Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the ex (show more...)Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain protein concentrations that vastly exceed those of the EV proteome and contain particles in the same size and density range (e.g. protein aggregates and apolipoprotein particles). Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion and precipitation methods. Here, we describe an approach based on 300 kDa MWCO filtration, which allows processing of multiple samples in parallel within a reasonable timeframe and at moderate cost. We demonstrate that our strategy is capable of quantitatively retaining EV particles on filters, whilst allowing extensive washing with relatively high percentages of the mild detergent TWEEN-20. In addition, we provide evidence that the retained EVs can be recuperated from the filter for qualitative studies or can be directly lysed on the filter for the recovery of the EV protein cargo for proteome analysis. Applying this strategy on MCF7 conditioned medium using different percentages of serum, we observed dramatic changes in the EV proteome. (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 Gag-EGFP expressing 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 Protein markers EV: None non-EV: Calnexin/ Albumin/ Argonaute-2/ PMP70/ Prohibitin/ Calreticulin/ GM130/ Tamm-Horsfall protein Proteomics yes Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 300 Membrane type Polyethersulfone (PES) Characterization: Protein analysis Protein Concentration Method Peptide concentration Western Blot Antibody details provided? No Not detected contaminants Calnexin Proteomics database No Detected contaminants Albumin/ Argonaute-2/ PMP70/ Prohibitin Not detected contaminants Calreticulin/ GM130/ Tamm-Horsfall protein Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 145 EV concentration Yes Particle yield particles per milliliter of starting sample: 6.50E+08

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