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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180079	2/2	Mus musculus	B16	(d)(U)C	Lucia Paolini	2020	67%
Study summary Full title Fourier-transform Infrared (FT-IR) spectroscopy fingerprints subpopulations of extracellular vesicles of different sizes and cellular origin All authors Lucia Paolini, Stefania Federici, Giovanni Consoli, Diletta Arceri, Annalisa Radeghieri, Ivano Alessandri, Paolo Bergese Journal J Extracell Vesicles Abstract Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the (show more...)Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the common strategy is based on searching and probing set of molecular components and physical properties intended to be univocally characteristics of the target subpopulation. Pitfalls include the risk to opt for an unsuitable marker set - which may either not represent the subpopulation or also cover other unintended subpopulations - and the need to use different characterization techniques and equipment. This approach focused on specific markers may result inadequate to routinely deal with EV subpopulations that have an intrinsic high level of heterogeneity. In this paper, we show that Fourier-transform Infrared (FT-IR) spectroscopy can provide a collective fingerprint of EV subpopulations in one single experiment. FT-IR measurements were performed on large (LEVs, ~600 nm), medium (MEVs, ~200 nm) and small (SEVs ~60 nm) EVs enriched from two different cell lines medium: murine prostate cancer (TRAMP-C2) and skin melanoma (B16). Spectral regions between 3100-2800 cm-1 and 1880-900 cm-1, corresponding to functional groups mainly ascribed to lipid and protein contributions, were acquired and processed by Principal Component Analysis (PCA). LEVs, MEVs and SEVs were separately grouped for both the considered cell lines. Moreover, subpopulations of the same size but from different sources were assigned (with different degrees of accuracy) to two different groups. These findings demonstrate that FT-IR has the potential to quickly fingerprint EV subpopulations as a whole, suggesting an appealing complement/alternative for their characterization and grading, extendable to healthy and pathological EVs and fully artificial nanovesicles. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: ADAM10/ Annexin V/ Flotillin1/ CD81 non-EV: GM130 Proteomics no Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells B16 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 1 Wash: time (min) 120 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Colorimetric Nanoplasmonic assay Western Blot Detected EV-associated proteins Flotillin1/ Annexin V/ ADAM10/ CD81 Not detected contaminants GM130 Characterization: Lipid analysis Yes EM EM-type Atomic force-EM Image type Close-up, Wide-field Report size (nm) large EVS 690 nm, medium Evs 230 nm, small Evs 50 nm

	EV180057	1/1	Rattus norvegicus	rBMSC	(d)(U)C	Gissi, Clarissa	2020	67%
Study summary Full title Extracellular vesicles from rat-bone-marrow mesenchymal stromal/stem cells improve tendon repair in rat Achilles tendon injury model in dose-dependent manner: A pilot study All authors Clarissa Gissi, Annalisa Radeghieri, Cristina Antonetti Lamorgese Passeri, Marialucia Gallorini, Lucia Calciano, Francesco Oliva, Francesca Veronesi, Andrea Zendrini, Amelia Cataldi, Paolo Bergese, Nicola Maffulli, Anna Concetta Berardi Journal PLoS One Abstract Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely med (show more...)Mesenchymal stromal/stem cells (MSCs) are increasingly employed for tissue regeneration, largely mediated through paracrine actions. Currently, extracellular vesicles (EVs) released by MSCs are major mediators of these paracrine effects. We evaluated whether rat-bone-marrow-MSC-derived EVs (rBMSCs-EVs) can ameliorate tendon injury in an in vivo rat model. Pro-collagen1A2 and MMP14 protein are expressed in rBMSC-EVs, and are important factors for extracellular-matrix tendon-remodeling. In addition, we found pro-collagen1A2 in rBMSC-EV surface-membranes by dot blot. In vitro on cells isolated from Achilles tendons, utilized as rBMSC -EVs recipient cells, EVs at both low and high doses induce migration of tenocytes; at higher concentration, they induce proliferation and increase expression of Collagen type I in tenocytes. Pretreatment with trypsin abrogate the effect of EVs on cell proliferation and migration, and the expression of collagen I. When either low- or high-dose rBMSCs-EVs were injected into a rat-Achilles tendon injury-model (immediately after damage), at 30 days, rBMSC-EVs were found to have accelerated the remodeling stage of tendon repair in a dose-dependent manner. At histology and histomorphology evaluation, high doses of rBMSCs-EVs produced better restoration of tendon architecture, with optimal tendon-fiber alignment and lower vascularity. Higher EV-concentrations demonstrated greater expression of collagen type I and lower expression of collagen type III. BMSC-EVs hold promise as a novel cell-free modality for the management of tendon injuries. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ Pro-collagen1A2/ Annexin/ TERT/ Annexin V non-EV: Argonaute2/ GM130 Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells rBMSC EV-harvesting Medium Serum free medium Cell viability (%) 100 Cell count 4.18x10e6 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: time(min) 70 Pelleting: rotor type SW 28 Pelleting: speed (g) 120000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD81/ Annexin V/ TERT/ Pro-collagen1A2 Detected contaminants Argonaute2 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Atomic force-EM Image type Wide-field Report size (nm) 95 Report type Not Reported EV-concentration Yes Particle yield 1E11 per particles mL starting sample

	XL5296IL	1/2	Macaca mulatta	Cervicovaginal lavage fluid	(d)(U)C	Zhao, Zezhou	2020	66%
Study summary Full title miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential antiretroviral factor in macrophages All authors Zezhou Zhao, Dillon C Muth, Kathleen Mulka, Zhaohao Liao, Bonita H Powell, Grace V Hancock, Kelly A Metcalf Pate, Kenneth W Witwer Journal FEBS Open Bio Abstract Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage ( (show more...)Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract. (hide) EV-METRIC 66% (66th 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 Cervicovaginal lavage 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 (d)(U)C Adj. k-factor 113.6 (pelleting) Protein markers EV: CD81/ CD63 non-EV: calnexin Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Macaca mulatta Sample Type Cervicovaginal lavage fluid 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: time(min) 120 Pelleting: rotor type AH-650 Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 113.6 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD63, CD81 Not detected contaminants calnexin Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) xx-xxx EV concentration Yes Particle yield 2.5E7-3.5E9 EM EM-type Transmission-EM Image type Close-up Report size (nm) up to 200 Extra information Sample volumes and yield limited the isolation and characterization steps we were able to perform.

	XL5296IL	2/2	Macaca mulatta	Cervicovaginal lavage fluid	(d)(U)C	Zhao, Zezhou	2020	66%
Study summary Full title miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential antiretroviral factor in macrophages All authors Zezhou Zhao, Dillon C Muth, Kathleen Mulka, Zhaohao Liao, Bonita H Powell, Grace V Hancock, Kelly A Metcalf Pate, Kenneth W Witwer Journal FEBS Open Bio Abstract Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage ( (show more...)Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract. (hide) EV-METRIC 66% (66th 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 Cervicovaginal lavage fluid Sample origin SIVmac251-infected 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 (d)(U)C Adj. k-factor 113.6 (pelleting) Protein markers EV: CD81/ CD63 non-EV: calnexin Proteomics no Show all info Study aim Biomarker, Identification of content (omics approaches) Sample Species Macaca mulatta Sample Type Cervicovaginal lavage fluid 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: time(min) 120 Pelleting: rotor type AH-650 Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 113.6 Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins CD63, CD81 Not detected contaminants calnexin Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) xx-xxx EV concentration Yes Particle yield 1.75E8-2.15E9 EM EM-type Transmission-EM Image type Close-up Report size (nm) up to 200 nm Extra information Samples from SIV Infected animals were pooled for WB analysis

	EV200025	3/4	Homo sapiens	Blood plasma	UF SEC (non-commercial) cation-exchange chromatography	Van Deun J	2020	63%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Spiked with ES2 EVs 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 Size-exclusion chromatography (non-commercial) cation-exchange chromatography Protein markers EV: CD63 non-EV: ApoA1/ ApoB100 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Other Name other separation method cation-exchange chromatography Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants ApoA1 ELISA Detected contaminants ApoB100 Characterization: Lipid analysis No Characterization: Particle analysis NTA EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200025	4/4	Homo sapiens	Blood plasma	UF SEC (non-commercial)	Van Deun J	2020	63%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Spiked with ES2 EVs 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD63 non-EV: ApoA1/ ApoB100 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants ApoA1 ELISA Detected contaminants ApoB100 Characterization: Lipid analysis No Characterization: Particle analysis NTA EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV240193	3/4	Homo sapiens	Blood plasma	UF SEC (non-commercial) cation-exchange chromatography	Van Deun J	2020	63%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Spiked with ES2 EVs 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 Size-exclusion chromatography (non-commercial) cation-exchange chromatography Protein markers EV: CD63 non-EV: ApoA1/ ApoB100 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Other Name other separation method cation-exchange chromatography Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants ApoA1 ELISA Detected contaminants ApoB100 Characterization: Lipid analysis No Characterization: Particle analysis NTA EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV240193	4/4	Homo sapiens	Blood plasma	UF SEC (non-commercial)	Van Deun J	2020	63%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Spiked with ES2 EVs 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 Size-exclusion chromatography (non-commercial) Protein markers EV: CD63 non-EV: ApoA1/ ApoB100 Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Ultra filtration Cut-off size (kDa) 10 Membrane type Regenerated cellulose Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Resin type Sepharose CL-4B Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63 Detected contaminants ApoA1 ELISA Detected contaminants ApoB100 Characterization: Lipid analysis No Characterization: Particle analysis NTA EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200073	1/4	Homo sapiens	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON)	Bordas, Marie	2020	63%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 63% (25th 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 Solid Tissue Sample origin CLL Focus vesicles exosome 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 (d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON) Protein markers EV: TSG101/ CD81/ Flotillin1/ CD9 non-EV: Cytochrome C/ GM130/ Calreticulin Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) Other Name other separation method SEC (IZON) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Flotillin1/ CD9/ TSG101/ CD81 Detected contaminants Calreticulin Not detected contaminants Cytochrome C/ GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 150 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 4.00E+10 EM EM-type Immuno-EM/ Transmission-EM EM protein Other;HLA-DR Image type Close-up

	EV200073	3/4	Mus musculus	Solid Tissue	(d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON)	Bordas, Marie	2020	63%
Study summary Full title Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues All authors Marie Bordas, Géraldine Genard, Sibylle Ohl, Michelle Nessling, Karsten Richter, Tobias Roider, Sascha Dietrich, Kendra K Maaß, Martina Seiffert Journal Int J Mol Sci Abstract Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication rel (show more...)Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. (hide) EV-METRIC 63% (25th 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 Solid Tissue Sample origin CLL Focus vesicles exosome 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 (d)(U)C Size-exclusion chromatopraphy (IZON) DC SEC (IZON) Protein markers EV: Alix/ TSG101/ CD81 non-EV: ATPA5/ Calreticulin Proteomics no Show all info Study aim Function/New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Solid Tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed No Density cushion Density medium Sucrose Other Name other separation method Size-exclusion chromatopraphy (IZON) Other Name other separation method SEC (IZON) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins Alix/ TSG101/ CD81 Detected contaminants Calreticulin Not detected contaminants ATPA5 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 145 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.50E+10 EM EM-type Transmission-EM Image type Close-up

	EV200038	4/4	Homo sapiens	Blood plasma	CD31 MicroBead Kit	Prattichizzo, Francesco	2020	63%
Study summary Full title CD31 + Extracellular Vesicles From Patients With Type 2 Diabetes Shuttle a miRNA Signature Associated With Cardiovascular Complications All authors Francesco Prattichizzo, Valeria De Nigris, Jacopo Sabbatinelli, Angelica Giuliani, Carlos Castaño, Marcelina Párrizas, Isabel Crespo, Annalisa Grimaldi, Nicolò Baranzini, Rosangela Spiga, Elettra Mancuso, Maria Rita Rippo, Antonio Domenico Procopio, Anna Novials, Anna Rita Bonfigli, Silvia Garavelli, Lucia La Sala, Giuseppe Matarese, Paola de Candia, Fabiola Olivieri, Antonio Ceriello Journal Diabetes Abstract Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (show more...)Innovative biomarkers are needed to improve the management of patients with type 2 diabetes mellitus (T2DM). Blood circulating miRNAs have been proposed as a potential tool to detect T2DM complications, but the lack of tissue specificity, among other reasons, has hampered their translation to clinical settings. Extracellular vesicle (EV)-shuttled miRNAs have been proposed as an alternative approach. Here, we adapted an immunomagnetic bead-based method to isolate plasma CD31+ EVs to harvest vesicles deriving from tissues relevant for T2DM complications. Surface marker characterization showed that CD31+ EVs were also positive for a range of markers typical of both platelets and activated endothelial cells. After characterization, we quantified 11 candidate miRNAs associated with vascular performance and shuttled by CD31+ EVs in a large (n = 218) cross-sectional cohort of patients categorized as having T2DM without complications, having T2DM with complications, and control subjects. We found that 10 of the tested miRNAs are affected by T2DM, while the signature composed by miR-146a, -320a, -422a, and -451a efficiently identified T2DM patients with complications. Furthermore, another CD31+ EV-shuttled miRNA signature, i.e., miR-155, -320a, -342-3p, -376, and -422a, detected T2DM patients with a previous major adverse cardiovascular event. Many of these miRNAs significantly correlate with clinical variables held to play a key role in the development of complications. In addition, we show that CD31+ EVs from patients with T2DM are able to promote the expression of selected inflammatory mRNAs, i.e., CCL2, IL-1α, and TNFα, when administered to endothelial cells in vitro. Overall, these data suggest that the miRNA cargo of plasma CD31+ EVs is largely affected by T2DM and related complications, encouraging further research to explore the diagnostic potential and the functional role of these alterations. (hide) EV-METRIC 63% (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 Blood plasma Sample origin Type 2 Diabetes 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 CD31 MicroBead Kit Protein markers EV: TSG101/ CD63/ CD81/ CD31/ CD9/ Alix non-EV: ApoA1 Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method CD31 MicroBead Kit Selected surface protein(s) CD31 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Alix/ CD31/ CD63/ TSG101 Not detected contaminants ApoA1 Flow cytometry specific beads Detected EV-associated proteins CD31/ CD9/ CD63/ CD81 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV200042	2/2	Mus musculus	Immortalized bone marrow-derived macrophages (iBMDMs)	DG (d)(U)C DC Filtration	Laura Bouchareychas	2020	63%
Study summary Full title Macrophage Exosomes Resolve Atherosclerosis by Regulating Hematopoiesis and Inflammation via MicroRNA Cargo All authors Laura Bouchareychas, Phat Duong, Sergio Covarrubias, Eric Alsop, Tuan Anh Phu, Allen Chung, Michael Gomes, David Wong, Bessie Meechoovet, Allyson Capili, Ryo Yamamoto, Hiromitsu Nakauchi, Michael T McManus, Susan Carpenter, Kendall Van Keuren-Jensen, Robert L Raffai Journal Cell Rep Abstract Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remai (show more...)Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery. (hide) EV-METRIC 63% (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 genetically modified cell line Focus vesicles exosome 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 DG (d)(U)C DC Filtration Protein markers EV: Alix/ Flotillin1 non-EV: Calnexin/ GM130 Proteomics no EV density (g/ml) 1.09 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Immortalized bone marrow-derived macrophages (iBMDMs) EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 95 Cell count 3.50E+07 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed 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) 12 Sample volume (mL) 3 Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.22µm or 0.2µm Density cushion Density medium Iodixanol Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Detected EV-associated proteins Flotillin1/ Alix Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type Other;RNase A/T1 Mix RNAse concentration 0.4 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 74.58 EV concentration Yes Particle yield Yes, as number of particles per million cells 2.40E+09

	EV200037	1/2	Homo sapiens	HLA-DR15+ B cells	DG SEC (non-commercial) PEG precipitation	Xiaogang Zhang	2020	63%
Study summary Full title A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity All authors Xiaogang Zhang , Ellen G. F. Borg , A. Manuel Liaci , Harmjan R. Vos & Willem Stoorvogel Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellul (show more...)Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment. (hide) EV-METRIC 63% (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 Density gradient Size-exclusion chromatography (non-commercial) PEG precipitation Protein markers EV: CD81/ MHC2/ CD63 non-EV: None Proteomics no EV density (g/ml) 1.09-1.13 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HLA-DR15+ B cells EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) 98 Separation Method Density gradient Type Continuous Lowest density fraction 0% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 60 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 0.3 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.6 Resin type Sepharose CL-2B Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ MHC2/ CD81 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV200037	2/2	Homo sapiens	Blood plasma	DG SEC (non-commercial) PEG precipitation	Xiaogang Zhang	2020	63%
Study summary Full title A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity All authors Xiaogang Zhang , Ellen G. F. Borg , A. Manuel Liaci , Harmjan R. Vos & Willem Stoorvogel Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellul (show more...)Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >105-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of ill-defined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment. (hide) EV-METRIC 63% (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 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 Density gradient Size-exclusion chromatography (non-commercial) PEG precipitation Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics yes EV density (g/ml) 1.09-1.13 Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method Density gradient Type Continuous Lowest density fraction 0% Highest density fraction 60% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 2 Orientation Bottom-up Rotor type SW 60 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 0.3 Fraction processing Size-exclusion chromatography Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.6 Resin type Sepharose CL-2B Other Name other separation method PEG precipitation Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins CD9/ CD63/ CD81 Proteomics database Yes: Characterization: Lipid analysis No EM EM-type Transmission-EM/ Cryo-EM Image type Wide-field

	EV200011	1/1	Bos taurus	milk	(d)(U)C UF	Lingjun, Tong	2020	63%
Study summary Full title Oral Administration of Bovine Milk-Derived Extracellular Vesicles Alters the Gut Microbiota and Enhances Intestinal Immunity in Mice All authors Lingjun Tong, Haining Hao, Xinyi Zhang, Zhe Zhang, Youyou Lv, Lanwei Zhang, Huaxi Yi Journal Mol Nutr Food Res Abstract Scope: Milk-derived extracellular vesicles (mEVs) as nanoparticles are being developed as novel drug (show more...)Scope: Milk-derived extracellular vesicles (mEVs) as nanoparticles are being developed as novel drug vehicles due to their pivotal role in cell-cell communication. As an important bioactive component in milk, little is known about their effect on the gut microbiota and intestinal immunity. Therefore, the effects of mEVs on gut microbiota and intestinal immunity in mice are investigated. Methods and results: First, a new method to obtain high-yield mEVs is developed. Afterward, the colonic contents from C57BL/6 mice fed different doses of mEVs (8 weeks) are collected and the microbial composition via 16S rRNA gene sequencing is analyzed. It is found that mEVs could alter the gut microbiota composition and modulate their metabolites-short-chain fatty acids (SCFAs). Furthermore, the effects of mEVs on intestinal immunity are evaluated. It is observed that the expression levels of Muc2, RegIIIγ, Myd88, GATA4 genes, and IgA, sIgA are increased in the intestine, which are significant for the integrity of the mucus layer. Conclusion: These findings reveal that the genes with critical importance for intestinal barrier function and immune regulation are modified in mice by oral administration mEVs, which also result in the changes of the relative composition of gut microbiome and SCFAs. (hide) EV-METRIC 63% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type milk Sample origin 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 (d)(U)C UF Protein markers EV: TSG101/ Alix/ CD9/ CD81 non-EV: Calnexin/ Histone H3 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Characterization: Protein analysis PMID previous EV protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD9/ TSG101/ Alix/ CD81 Not detected contaminants Calnexin/ Histone H3 Flow cytometry aspecific beads Detected EV-associated proteins CD9 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis Extra particle analysis NTA Report type Mean Reported size (nm) 102.8 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 1.10E+10 EM EM-type Transmission-EM Image type Close-up

	EV190050	1/1	Mus musculus	4T1	DG Filtration SEC SEC (non-commercial)	Ger J A Arkesteijn	2020	63%
Study summary Full title Improved Flow Cytometric Light Scatter Detection of Submicron-Sized Particles by Reduction of Optical Background Signals All authors Ger J A Arkesteijn, Estefanía Lozano-Andrés, Sten F W M Libregts, Marca H M Wauben Journal Cytometry A Abstract Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of (show more...) Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron-sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV-related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV-subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC-detection of submicron-sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron-sized particles. We found that a combination of an 8-mm obscuration bar and a 200-μm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100-nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron-sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron-sized particles. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. (hide) EV-METRIC 63% (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 breast tumor model 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 DG Filtration SEC Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ Flotillin2/ HSP70/ MHC2/ CD9/ MHC1 non-EV: Proteomics no EV density (g/ml) 1.10-1.12 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 90 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1091 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Filtration steps 0.45µm > x > 0.22µm, Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Other Name other separation method Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ CD63 Not detected EV-associated proteins HSP90/ HSP70/ MHC1/ CD81/ Flotillin1/ TSG101/ MHC2/ Flotillin2/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 139.8 EV concentration Yes

	EV190036	1/3	Homo sapiens	OX1-19	Filtration qEV	Hicks, David	2020	63%
Study summary Full title Extracellular Vesicles Isolated from Human Induced Pluripotent Stem Cell-Derived Neurons Contain a Transcriptional Network All authors David A Hicks, Alys C Jones, Nicola J Corbett, Kate Fisher, Stuart M Pickering-Brown, Mark P Ashe, Nigel M Hooper Journal Neurochem Res. Abstract Healthy brain function is mediated by several complementary signalling pathways, many of which are d (show more...)Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30–100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons. (hide) EV-METRIC 63% (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 Filtration qEV Protein markers EV: TSG101/ CD9 non-EV: Mitofilin/ Grp78 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells OX1-19 EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ TSG101 Not detected contaminants Mitofilin/ Grp78 Proteomics database No Characterization: RNA analysis RNA analysis Type RNA sequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.05 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) SEC fraction-dependent EM EM-type Transmission-EM Image type Wide-field

	EV190036	2/3	Homo sapiens	OX1-19	Filtration qEV	Hicks, David	2020	63%
Study summary Full title Extracellular Vesicles Isolated from Human Induced Pluripotent Stem Cell-Derived Neurons Contain a Transcriptional Network All authors David A Hicks, Alys C Jones, Nicola J Corbett, Kate Fisher, Stuart M Pickering-Brown, Mark P Ashe, Nigel M Hooper Journal Neurochem Res. Abstract Healthy brain function is mediated by several complementary signalling pathways, many of which are d (show more...)Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30–100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons. (hide) EV-METRIC 63% (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 Filtration qEV Protein markers EV: TSG101/ CD9 non-EV: Mitofilin/ Grp78 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells OX1-19 EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ TSG101 Not detected contaminants Grp78/ Mitofilin Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.05 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) SEC fraction-dependent EM EM-type Transmission-EM Image type Wide-field

	EV190036	3/3	Homo sapiens	OX1-19	Filtration qEV	Hicks, David	2020	63%
Study summary Full title Extracellular Vesicles Isolated from Human Induced Pluripotent Stem Cell-Derived Neurons Contain a Transcriptional Network All authors David A Hicks, Alys C Jones, Nicola J Corbett, Kate Fisher, Stuart M Pickering-Brown, Mark P Ashe, Nigel M Hooper Journal Neurochem Res. Abstract Healthy brain function is mediated by several complementary signalling pathways, many of which are d (show more...)Healthy brain function is mediated by several complementary signalling pathways, many of which are driven by extracellular vesicles (EVs). EVs are heterogeneous in both size and cargo and are constitutively released from cells into the extracellular milieu. They are subsequently trafficked to recipient cells, whereupon their entry can modify the cellular phenotype. Here, in order to further analyse the mRNA and protein cargo of neuronal EVs, we isolated EVs by size exclusion chromatography from human induced pluripotent stem cell (iPSC)-derived neurons. Electron microscopy and dynamic light scattering revealed that the isolated EVs had a diameter of 30–100 nm. Transcriptomic and proteomics analyses of the EVs and neurons identified key molecules enriched in the EVs involved in cell surface interaction (integrins and collagens), internalisation pathways (clathrin- and caveolin-dependent), downstream signalling pathways (phospholipases, integrin-linked kinase and MAPKs), and long-term impacts on cellular development and maintenance. Overall, we show that key signalling networks and mechanisms are enriched in EVs isolated from human iPSC-derived neurons. (hide) EV-METRIC 63% (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 Filtration qEV Protein markers EV: TSG101/ CD9 non-EV: Mitofilin/ Grp78 Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells OX1-19 EV-harvesting Medium Serum free medium Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD9/ TSG101 Not detected contaminants Mitofilin/ Grp78 Proteomics database No Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.05 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) SEC fraction-dependent EM EM-type Transmission-EM Image type Wide-field

	EV190013	1/1	Homo sapiens	UW228-2	(d)(U)C Filtration	Johnson, Suzanne M	2020	63%
Study summary Full title Large Extracellular Vesicles Can be Characterised by Multiplex Labelling Using Imaging Flow Cytometry All authors Suzanne M Johnson, Antonia Banyard, Christopher Smith, Aleksandr Mironov, Martin G McCabe Journal Int J Mol Sci Abstract Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, pro (show more...)Extracellular vesicles (EVs) are heterogeneous in size (30 nm-10 µm), content (lipid, RNA, DNA, protein), and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs from medulloblastoma cells and examine EV marker expression and diameter using imaging flow cytometry. Our approach permits the characterisation of each large EVs as an individual event, decorated with multiple fluorescently conjugated markers with the added advantage of visualising each event to ensure robust gating strategies are applied. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and tunable resistance pulse sensing (TRPS). Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed. Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions: We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9, and LAMP1) on individual EVs within a widely heterogeneous population. Taken together, data presented here strongly support the value of exploring large EVs in clinical samples for potential biomarkers, useful in diagnostic screening and disease monitoring. (hide) EV-METRIC 63% (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 (d)(U)C Filtration Protein markers EV: CD63/ CD9/ LAMP1 non-EV: Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UW228-2 EV-harvesting Medium Serum free medium Cell viability (%) NA 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: time(min) 30 Pelleting: rotor type A-4-38 Pelleting: speed (g) 2000 Filtration steps > 0.45 µm, Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Imagestream Detected EV-associated proteins CD63 Detected EV-associated proteins CD63/ CD9/ LAMP1 Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 600-6000 Particle analysis: flow cytometry Flow cytometer type Imagestream Hardware adjustment EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 4500

	EV200192	1/2	Exophiala dermatitidis	EXF-10123	(d)(U)C Filtration UF DG	Lavrin, Teja	2020	58%
Study summary Full title The Neurotropic Black Yeast Exophiala dermatitidis Induces Neurocytotoxicity in Neuroblastoma Cells and Progressive Cell Death All authors Teja Lavrin, Tilen Konte, Rok Kostanjšek, Simona Sitar, Kristina Sepčič, Sonja Prpar Mihevc, Ema Žagar, Vera Župunski, Metka Lenassi, Boris Rogelj, Nina Gunde Cimerman Journal Cells Abstract The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits d (show more...)The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of E. dermatitidis on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of E. dermatitidis biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of E. dermatitidis with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by E. dermatitidis hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's. (hide) EV-METRIC 58% (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 Filtration Ultrafiltration Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.09–1.32 Show all info Study aim Function Sample Species Exophiala dermatitidis Sample Type Cell culture supernatant EV-producing cells EXF-10123 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type MLA-50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 20 Highest density fraction 60 Total gradient volume, incl. sample (mL) 4.8 Sample volume (mL) 0.4 Orientation Not specified Rotor type Not specified Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.4 Fraction processing Precipitation Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Other Name other separation method Filtration Other Name other separation method Ultrafiltration Other Name other separation method Density gradient Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report type Mean Report size 90 EV-concentration Yes

	EV200192	2/2	Exophiala dermatitidis	EXF-10123	(d)(U)C Filtration UF DG	Lavrin, Teja	2020	58%
Study summary Full title The Neurotropic Black Yeast Exophiala dermatitidis Induces Neurocytotoxicity in Neuroblastoma Cells and Progressive Cell Death All authors Teja Lavrin, Tilen Konte, Rok Kostanjšek, Simona Sitar, Kristina Sepčič, Sonja Prpar Mihevc, Ema Žagar, Vera Župunski, Metka Lenassi, Boris Rogelj, Nina Gunde Cimerman Journal Cells Abstract The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits d (show more...)The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of E. dermatitidis on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of E. dermatitidis biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of E. dermatitidis with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by E. dermatitidis hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's. (hide) EV-METRIC 58% (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 Melanin inhibitor 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 Density gradient Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) 1.09–1.32 Show all info Study aim Function Sample Species Exophiala dermatitidis Sample Type Cell culture supernatant EV-producing cells EXF-10123 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type MLA-50 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type TLA-55 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 20 Highest density fraction 60 Total gradient volume, incl. sample (mL) 4.8 Sample volume (mL) 0.4 Orientation Not specified Rotor type Not specified Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.4 Fraction processing Precipitation Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Other Name other separation method Filtration Other Name other separation method Ultrafiltration Other Name other separation method Density gradient Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis Particle analysis: flow cytometry Hardware adjustment EM EM-type Transmission-EM Image type Close-up Report type Mean Report size 75 EV-concentration Yes

	EV200025	1/4	Homo sapiens	ES2	(d)(U)C DG UF	Van Deun J	2020	57%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 57% (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 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 Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ES2 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Top-down Rotor type SW27.1 Ti 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 Resin type Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV200025	2/4	Homo sapiens	Gli36 EGFRvIII	(d)(U)C DG UF	Van Deun J	2020	57%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 57% (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 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 Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Gli36 EGFRvIII EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Top-down Rotor type SW27.1 Ti 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 Resin type Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240193	1/4	Homo sapiens	ES2	(d)(U)C DG UF	Van Deun J	2020	57%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 57% (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 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 Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells ES2 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Top-down Rotor type SW27.1 Ti 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 Resin type Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV240193	2/4	Homo sapiens	Gli36 EGFRvIII	(d)(U)C DG UF	Van Deun J	2020	57%
Study summary Full title Integrated Dual-Mode Chromatography to Enrich Extracellular Vesicles from Plasma. All authors Van Deun J, Jo A, Li H, Lin HY, Weissleder R, Im H, Lee H Journal Adv Biosyst Abstract Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzin (show more...)Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 -fold. Given their overlap in size, LPPs cannot be completely removed using standard size-exclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single column device: i) size exclusion to remove high-density lipoproteins (HDLs) that are smaller than EVs/ and ii) cation exchange to clear positively charged ApoB100-containing LPPs, mostly (very) low-density lipoproteins (V)LDLs, from negatively charged EVs. The strategy enables DMC to deplete most LPPs (>97% of HDLs and >99% of (V)LDLs) from human plasma, while retaining EVs (>30% of input). Furthermore, the two-in-one operation is fast (15 min per sample) and equipment-free. With abundant LPPs removed, DMC-prepared samples facilitate EV identification in imaging analyses and improve the accuracy for EV protein analysis. (hide) EV-METRIC 57% (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 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 Protein markers EV: CD9/ CD63/ CD81 non-EV: None Proteomics no EV density (g/ml) 1.1 Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Gli36 EGFRvIII EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Continuous Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 1 Orientation Top-down Rotor type SW27.1 Ti 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 Resin type Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry specific beads Detected EV-associated proteins CD9/ CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230401	3/24	Pseudomonas aeruginosa	PAO1	(d)(U)C Filtration	Zhang L	2020	57%
Study summary Full title Proteomic Analysis of Vesicle-Producing PAO1 Exposed to X-Ray Irradiation. All authors Zhang L, Zhao SQ, Zhang J, Sun Y, Xie YL, Liu YB, Ma CC, Jiang BG, Liao XY, Li WF, Cheng XJ, Wang ZL Journal Front Microbiol Abstract Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destructi (show more...)Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destruction. However, how pathogens respond to irradiation stress has not yet been fully elucidated. Here, we observed that PAO1 could release nucleic acids into the extracellular environment under X-ray irradiation. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), X-ray irradiation was observed to induce outer membrane vesicle (OMV) formation in PAO1. The size distribution of the OMVs of the irradiated PAO1 was similar to that of the OMVs of the non-irradiated PAO1 according to nanoparticle tracking analysis (NTA). The pyocin-related proteins are involved in OMV production in PAO1 under X-ray irradiation conditions, and that this is regulated by the key SOS gene . The OMV production was significantly impaired in the irradiated PAO1 Δ mutant, suggesting that Lys endolysin is associated with OMV production in PAO1 upon irradiation stress. Meanwhile, no significant difference in OMV production was observed between PAO1 lacking the , , or genes and the parent strain, demonstrating that the irradiation-induced OMV biosynthesis of was independent of the quinolone signal (PQS). (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin Control condition Focus vesicles Outer membrane vesicle (OMV) 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: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant EV-producing cells PAO1 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes Particle yield as number of particles per million colony-forming units: 5.00E+11 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230401	4/24	Pseudomonas aeruginosa	PAO1	(d)(U)C Filtration	Zhang L	2020	57%
Study summary Full title Proteomic Analysis of Vesicle-Producing PAO1 Exposed to X-Ray Irradiation. All authors Zhang L, Zhao SQ, Zhang J, Sun Y, Xie YL, Liu YB, Ma CC, Jiang BG, Liao XY, Li WF, Cheng XJ, Wang ZL Journal Front Microbiol Abstract Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destructi (show more...)Ionizing irradiation kills pathogens by destroying nucleic acids without protein structure destruction. However, how pathogens respond to irradiation stress has not yet been fully elucidated. Here, we observed that PAO1 could release nucleic acids into the extracellular environment under X-ray irradiation. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), X-ray irradiation was observed to induce outer membrane vesicle (OMV) formation in PAO1. The size distribution of the OMVs of the irradiated PAO1 was similar to that of the OMVs of the non-irradiated PAO1 according to nanoparticle tracking analysis (NTA). The pyocin-related proteins are involved in OMV production in PAO1 under X-ray irradiation conditions, and that this is regulated by the key SOS gene . The OMV production was significantly impaired in the irradiated PAO1 Δ mutant, suggesting that Lys endolysin is associated with OMV production in PAO1 upon irradiation stress. Meanwhile, no significant difference in OMV production was observed between PAO1 lacking the , , or genes and the parent strain, demonstrating that the irradiation-induced OMV biosynthesis of was independent of the quinolone signal (PQS). (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin irradiated Focus vesicles Outer membrane vesicle (OMV) 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: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant EV-producing cells PAO1 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 Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Filtration steps Between 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 170 EV concentration Yes Particle yield as number of particles per million colony-forming units: 5.50E+12 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230381	1/1	Filifactor alocis	ATCC 35896	(d)(U)C DG Filtration UF	Kim HY	2020	57%
Study summary Full title Characterization and immunostimulatory activity of extracellular vesicles from Filifactor alocis. All authors Kim HY, Lim Y, An SJ, Choi BK Journal Mol Oral Microbiol Abstract Filifactor alocis, a gram-positive, obligate anaerobic rod, is an emerging periodontal pathogen that (show more...)Filifactor alocis, a gram-positive, obligate anaerobic rod, is an emerging periodontal pathogen that is frequently isolated from patients with periodontitis, peri-implantitis, and apical periodontitis. Recent studies have shown that extracellular vesicles (EVs) from gram-negative periodontal pathogens, so-called outer membrane vesicles (OMVs), harbor various effector molecules responsible for inducing host inflammatory responses. However, there are no reports of EVs from F. alocis. In this study, we purified and characterized the protein profiles of EVs from F. alocis and investigated their immunostimulatory activity on human monocytic THP-1 and human oral keratinocyte HOK-16B cell lines. Highly pure EVs were obtained from F. alocis using density gradient ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy showed that F. alocis EVs were between 50 and 270 nm in diameter. Proteome analysis identified 28 proteins, including lipoproteins, autolysins, F. alocis complement inhibitor (FACIN), transporter-related proteins, metabolism-related proteins, and ribosomal proteins. Human cytokine array analysis showed that F. alocis EVs remarkably induced the expression of CCL1, CCL2, MIP-1, CCL5, CXCL1, CXCL10, ICAM-1, IL-1β, IL-1ra, IL-6, IL-8, MIF, SerpinE, and TNF-α in THP-1 cells and CXCL1, G-CSF, GM-CSF, IL-6, and IL-8 in HOK-16B cells. The immunostimulatory activity of F. alocis EVs was similar to that of the whole bacterial cells. Our findings provide new insight into the role of EVs from gram-positive oral bacteria in periodontal diseases. (hide) EV-METRIC 57% (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 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 Filtration Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Filifactor alocis Sample Type Cell culture supernatant EV-producing cells ATCC 35896 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type P70AT rotor Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 10% Highest density fraction 40% Orientation Bottom-up Speed (g) 160000 Duration (min) 240 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: speed (g) 100000 Pelleting: adjusted k-factor TDB Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) per million cells Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-270 EV concentration Yes Particle yield number of particles per million cells: 1.10E+13 EM EM-type Transmission-EM Image type Wide-field

	EV230334	1/2	Cystobacter ferrugineus	Cbfe23	(d)(U)C SEC (non-commercial) Filtration	Goes A	2020	57%
Study summary Full title Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections. All authors Goes A, Lapuhs P, Kuhn T, Schulz E, Richter R, Panter F, Dahlem C, Koch M, Garcia R, Kiemer AK, Müller R, Fuhrmann G Journal Cells Abstract In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annual (show more...)In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is , a bacterium that can invade and survive within mammalian cells. intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria strain Cbv34 and strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular . We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular . In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections. (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin Control condition Focus vesicles Other/ Outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Cystobacter ferrugineus Sample Type Cell culture supernatant EV-producing cells Cbfe23 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 100000 Filtration steps NS Size-exclusion chromatography Total column volume (mL) 60 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 100-350 NTA Report type Size range/distribution Reported size (nm) 50-250 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV230302	1/5	Staphylococcus aureus	O46	(d)(U)C DG UF Filtration	Tartaglia NR	2020	57%
Study summary Full title Extracellular vesicles produced by human and animal Staphylococcus aureus strains share a highly conserved core proteome. All authors Tartaglia NR, Nicolas A, Rodovalho VR, Luz BSRD, Briard-Bion V, Krupova Z, Thierry A, Coste F, Burel A, Martin P, Jardin J, Azevedo V, Le Loir Y, Guédon E Journal Sci Rep Abstract Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extr (show more...)Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches. (hide) EV-METRIC 57% (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 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells O46 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Density gradient Lowest density fraction 8% Highest density fraction 68% Orientation Top-down Speed (g) 100000 Duration (min) 150 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 170 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 170

	EV230302	2/5	Staphylococcus aureus	O11	(d)(U)C DG UF Filtration	Tartaglia NR	2020	57%
Study summary Full title Extracellular vesicles produced by human and animal Staphylococcus aureus strains share a highly conserved core proteome. All authors Tartaglia NR, Nicolas A, Rodovalho VR, Luz BSRD, Briard-Bion V, Krupova Z, Thierry A, Coste F, Burel A, Martin P, Jardin J, Azevedo V, Le Loir Y, Guédon E Journal Sci Rep Abstract Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extr (show more...)Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches. (hide) EV-METRIC 57% (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 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells O11 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Density gradient Lowest density fraction 8% Highest density fraction 68% Orientation Top-down Speed (g) 100000 Duration (min) 150 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120-130 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120-130

	EV230302	3/5	Staphylococcus aureus	RF122	(d)(U)C DG UF Filtration	Tartaglia NR	2020	57%
Study summary Full title Extracellular vesicles produced by human and animal Staphylococcus aureus strains share a highly conserved core proteome. All authors Tartaglia NR, Nicolas A, Rodovalho VR, Luz BSRD, Briard-Bion V, Krupova Z, Thierry A, Coste F, Burel A, Martin P, Jardin J, Azevedo V, Le Loir Y, Guédon E Journal Sci Rep Abstract Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extr (show more...)Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches. (hide) EV-METRIC 57% (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 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells RF122 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Density gradient Lowest density fraction 8% Highest density fraction 68% Orientation Top-down Speed (g) 100000 Duration (min) 150 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120-130 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120-130

	EV230302	4/5	Staphylococcus aureus	MW2. S. aureus N305	(d)(U)C DG UF Filtration	Tartaglia NR	2020	57%
Study summary Full title Extracellular vesicles produced by human and animal Staphylococcus aureus strains share a highly conserved core proteome. All authors Tartaglia NR, Nicolas A, Rodovalho VR, Luz BSRD, Briard-Bion V, Krupova Z, Thierry A, Coste F, Burel A, Martin P, Jardin J, Azevedo V, Le Loir Y, Guédon E Journal Sci Rep Abstract Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extr (show more...)Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches. (hide) EV-METRIC 57% (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 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells MW2. S. aureus N305 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Density gradient Lowest density fraction 8% Highest density fraction 68% Orientation Top-down Speed (g) 100000 Duration (min) 150 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120-130 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120-130

	EV230302	5/5	Staphylococcus aureus	Newbould 305	(d)(U)C DG UF Filtration	Tartaglia NR	2020	57%
Study summary Full title Extracellular vesicles produced by human and animal Staphylococcus aureus strains share a highly conserved core proteome. All authors Tartaglia NR, Nicolas A, Rodovalho VR, Luz BSRD, Briard-Bion V, Krupova Z, Thierry A, Coste F, Burel A, Martin P, Jardin J, Azevedo V, Le Loir Y, Guédon E Journal Sci Rep Abstract Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extr (show more...)Staphylococcus aureus is an important opportunistic pathogen of humans and animals. It produces extracellular vesicles (EVs) that are involved in cellular communication and enable inter-kingdom crosstalk, the delivery of virulence factors and modulation of the host immune response. The protein content of EVs determines their biological functions. Clarifying which proteins are selected, and how, is of crucial value to understanding the role of EVs in pathogenesis and the development of molecular delivery systems. Here, we postulated that S. aureus EVs share a common proteome containing components involved in cargo sorting. The EV proteomes of five S. aureus strains originating from human, bovine, and ovine hosts were characterised. The clustering of EV proteomes reflected the diversity of the producing strains. A total of 253 proteins were identified, 119 of which composed a core EV proteome with functions in bacterial survival, pathogenesis, and putatively in EV biology. We also identified features in the sequences of EV proteins and the corresponding genes that could account for their packaging into EVs. Our findings corroborate the hypothesis of a selective sorting of proteins into EVs and offer new perspectives concerning the roles of EVs in S. aureus pathogenesis in specific host niches. (hide) EV-METRIC 57% (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 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 Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Staphylococcus aureus Sample Type Cell culture supernatant EV-producing cells Newbould 305 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: speed (g) 150000 Density gradient Lowest density fraction 8% Highest density fraction 68% Orientation Top-down Speed (g) 100000 Duration (min) 150 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 120-130 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120-130

	EV230298	1/3	Escherichia coli	APEC O1	DG (d)(U)C UF Filtration	Hu R	2020	57%
Study summary Full title Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC). All authors Hu R, Li J, Zhao Y, Lin H, Liang L, Wang M, Liu H, Min Y, Gao Y, Yang M Journal Microb Cell Fact Abstract The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its nu (show more...)The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin Control condition Focus vesicles Other/ Outer membrane 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 Density gradient (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells APEC O1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 10% Highest density fraction 55% Speed (g) 180000 Duration (min) 960 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-200 EM EM-type Transmission-EM/ Scanning-EM Image type Wide-field

	EV230298	2/3	Escherichia coli	APEC O2	DG (d)(U)C UF Filtration	Hu R	2020	57%
Study summary Full title Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC). All authors Hu R, Li J, Zhao Y, Lin H, Liang L, Wang M, Liu H, Min Y, Gao Y, Yang M Journal Microb Cell Fact Abstract The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its nu (show more...)The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin Control condition Focus vesicles Other/ Outer membrane 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 Density gradient (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells APEC O2 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 10% Highest density fraction 55% Speed (g) 180000 Duration (min) 960 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230298	3/3	Escherichia coli	APEC 078	DG (d)(U)C UF Filtration	Hu R	2020	57%
Study summary Full title Exploiting bacterial outer membrane vesicles as a cross-protective vaccine candidate against avian pathogenic Escherichia coli (APEC). All authors Hu R, Li J, Zhao Y, Lin H, Liang L, Wang M, Liu H, Min Y, Gao Y, Yang M Journal Microb Cell Fact Abstract The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its nu (show more...)The well-known fact that avian pathogenic Escherichia coli (APEC) is harder to prevent due to its numerous serogroups has promoted the development of biological immunostimulatory materials as new vaccine candidates in poultry farms. Bacterial outer membrane vesicles (OMVs), known as spherical nanovesicles enriched with various immunostimulants, are naturally secreted by Gram-negative bacteria, and have gained much attention for developing effective vaccine candidates. Recent report has demonstrated that OMVs of APEC O78 can induce protective immunity in chickens. Here, a novel multi-serogroup OMVs (MOMVs) vaccine was developed to achieve cross-protection against APEC infection in broiler chickens. (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin Control condition Focus vesicles Other/ Outer membrane 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 Density gradient (Differential) (ultra)centrifugation Ultrafiltration Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Escherichia coli Sample Type Cell culture supernatant EV-producing cells APEC 078 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Lowest density fraction 10% Highest density fraction 55% Speed (g) 180000 Duration (min) 960 Fraction processing Centrifugation Pelleting: volume per fraction not spec Pelleting: duration (min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230285	1/2	Sus scrofa	Stool	(d)(U)C Filtration UF DG	Lagos L	2020	57%
Study summary Full title Isolation and Characterization of Extracellular Vesicles Secreted In Vitro by Porcine Microbiota. All authors Lagos L, Leanti La Rosa S, Ø Arntzen M, Ånestad R, Terrapon N, Gaby JC, Westereng B Journal Microorganisms Abstract The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic (show more...)The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic cells for intercellular communication, survival, and pathogenesis. Previous studies have illustrated the presence of EVs in supernatants from pure cultures of bacteria, including Gram-positive and Gram-negative glycan-degrading gut commensals. However, the isolation and characterization of EVs secreted by a complex microbial community have not been clearly reported. In a recent paper, we showed that wood-derived, complex β-mannan, which shares a structural similarity with conventional dietary fibers, can be used to modulate the porcine gut microbiota composition and activity. In this paper, we investigated the production, size, composition, and proteome of EVs secreted by pig fecal microbiota after 24 h enrichment on complex β-mannan. Using transmission electron microscopy and nanoparticle tracking analysis, we identified EVs with an average size of 165 nm. We utilized mass spectrometry-based metaproteomic profiling of EV proteins against a database of 355 metagenome-assembled genomes (MAGs) from the porcine colon and thereby identified 303 proteins. For EVs isolated from the culture grown on β-mannan, most proteins mapped to two MAGs, MAG53 and MAG272, belonging to the orders and , respectively. Furthermore, the MAG with the third-most-detected protein was MAG 343, belonging to the order . The most abundant proteins detected in the β-mannan EVs proteome were involved in translation, energy production, amino acid, and carbohydrate transport, as well as metabolism. Overall, this proof-of-concept study demonstrates the successful isolation of EVs released from a complex microbial community/ furthermore, the protein content of the EVs reflects the response of specific microbes to the available carbohydrate source. (hide) EV-METRIC 57% (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 Stool Sample origin No carbohydrate source 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 Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Stool 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 SW 50.1 Pelleting: speed (g) 114000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mode Reported size (nm) 105/ 350 EM EM-type Transmission-EM Image type Wide-field

	EV230285	2/2	Sus scrofa	Stool	(d)(U)C Filtration UF DG	Lagos L	2020	57%
Study summary Full title Isolation and Characterization of Extracellular Vesicles Secreted In Vitro by Porcine Microbiota. All authors Lagos L, Leanti La Rosa S, Ø Arntzen M, Ånestad R, Terrapon N, Gaby JC, Westereng B Journal Microorganisms Abstract The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic (show more...)The secretion of extracellular vesicles, EVs, is a common process in both prokaryotic and eukaryotic cells for intercellular communication, survival, and pathogenesis. Previous studies have illustrated the presence of EVs in supernatants from pure cultures of bacteria, including Gram-positive and Gram-negative glycan-degrading gut commensals. However, the isolation and characterization of EVs secreted by a complex microbial community have not been clearly reported. In a recent paper, we showed that wood-derived, complex β-mannan, which shares a structural similarity with conventional dietary fibers, can be used to modulate the porcine gut microbiota composition and activity. In this paper, we investigated the production, size, composition, and proteome of EVs secreted by pig fecal microbiota after 24 h enrichment on complex β-mannan. Using transmission electron microscopy and nanoparticle tracking analysis, we identified EVs with an average size of 165 nm. We utilized mass spectrometry-based metaproteomic profiling of EV proteins against a database of 355 metagenome-assembled genomes (MAGs) from the porcine colon and thereby identified 303 proteins. For EVs isolated from the culture grown on β-mannan, most proteins mapped to two MAGs, MAG53 and MAG272, belonging to the orders and , respectively. Furthermore, the MAG with the third-most-detected protein was MAG 343, belonging to the order . The most abundant proteins detected in the β-mannan EVs proteome were involved in translation, energy production, amino acid, and carbohydrate transport, as well as metabolism. Overall, this proof-of-concept study demonstrates the successful isolation of EVs released from a complex microbial community/ furthermore, the protein content of the EVs reflects the response of specific microbes to the available carbohydrate source. (hide) EV-METRIC 57% (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 Stool Sample origin ?-mannan 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 Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Sus scrofa Sample Type Stool 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 SW 50.1 Pelleting: speed (g) 114000 Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 4 Sample volume (mL) 1 Orientation Top-down Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mode Reported size (nm) 165/ 950 EM EM-type Transmission-EM Image type Close-up

	EV230273	2/4	Buttiauxella agrestis	JCM1090	DG (d)(U)C Filtration	Takaki K	2020	57%
Study summary Full title Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant. All authors Takaki K, Tahara YO, Nakamichi N, Hasegawa Y, Shintani M, Ohkuma M, Miyata M, Futamata H, Tashiro Y Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important (show more...)Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications. (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin TolB mutant Focus vesicles Other/ Outer membrane 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Buttiauxella agrestis Sample Type Cell culture supernatant EV-producing cells JCM1090 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes Translucent band Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 75 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-150

	EV230273	3/4	Buttiauxella agrestis	JCM1090	DG (d)(U)C Filtration	Takaki K	2020	57%
Study summary Full title Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant. All authors Takaki K, Tahara YO, Nakamichi N, Hasegawa Y, Shintani M, Ohkuma M, Miyata M, Futamata H, Tashiro Y Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important (show more...)Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications. (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin TolB mutant Focus vesicles Other/ Outer membrane 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Buttiauxella agrestis Sample Type Cell culture supernatant EV-producing cells JCM1090 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes Upper band Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 125 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-150

	EV230273	4/4	Buttiauxella agrestis	JCM1090	DG (d)(U)C Filtration	Takaki K	2020	57%
Study summary Full title Multilamellar and Multivesicular Outer Membrane Vesicles Produced by a Buttiauxella agrestis Mutant. All authors Takaki K, Tahara YO, Nakamichi N, Hasegawa Y, Shintani M, Ohkuma M, Miyata M, Futamata H, Tashiro Y Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important (show more...)Outer membrane vesicles (OMVs) are naturally released from Gram-negative bacteria and play important roles in various biological functions. Released vesicles are not uniform in shape, size, or characteristics, and little is known about this diversity of OMVs. Here, we show that deletion of , which encodes a part of the Tol-Pal system, leads to the production of multiple types of vesicles and increases overall vesicle production in the high-vesicle-forming type strain JCM 1090. The Δ mutant produced small OMVs and multilamellar/multivesicular OMVs (M-OMVs) as well as vesicles with a striking similarity to the wild type. M-OMVs, previously undescribed, contained triple-lamellar membrane vesicles and multiple vesicle-incorporating vesicles. Ultracentrifugation enabled the separation and purification of each type of OMV released from the Δ mutant, and visualization by quick-freeze deep-etch and replica electron microscopy indicated that M-OMVs are composed of several lamellar membranes. Visualization of intracellular compartments of Δ mutant cells showed that vesicles were accumulated in the broad periplasm, which is probably due to the low linkage between the outer and inner membranes attributed to the Tol-Pal defect. The outer membrane was invaginating inward by wrapping a vesicle, and the precursor of M-OMVs existed in the cell. Thus, we demonstrated a novel type of bacterial OMV and showed that unconventional processes enable the Δ mutant to form unique vesicles. Membrane vesicle (MV) formation has been recognized as a common mechanism in prokaryotes, and MVs play critical roles in intercellular interaction. However, a broad range of MV types and their multiple production processes make it difficult to gain a comprehensive understanding of MVs. In this work, using vesicle separation and electron microscopic analyses, we demonstrated that diverse types of outer membrane vesicles (OMVs) were released from an engineered strain, JCM 1090 Δ mutant. We also discovered a previously undiscovered type of vesicle, multilamellar/multivesicular outer membrane vesicles (M-OMVs), which were released by this mutant using unconventional processes. These findings have facilitated considerable progress in understanding MV diversity and expanding the utility of MVs in biotechnological applications. (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin TolB mutant Focus vesicles Other/ Outer membrane 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods Sample Species Buttiauxella agrestis Sample Type Cell culture supernatant EV-producing cells JCM1090 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) 10 Sample volume (mL) 1 Orientation Bottom-up Speed (g) 100000 Duration (min) 1200 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type P45AT Pelleting: speed (g) 150000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm EV-subtype Distinction between multiple subtypes Density Used subtypes Lower band Characterization: Protein analysis None Protein Concentration Method BCA Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 145 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-150

	EV230251	1/1	Avibacterium paragallinarum	hp8	(d)(U)C Filtration DG	Mei C	2020	57%
Study summary Full title Component Identification and Functional Analysis of Outer Membrane Vesicles Released by . All authors Mei C, Sun AH, Blackall PJ, Xian H, Li SF, Gong YM, Wang HJ Journal Front Microbiol Abstract , the causative agent of infectious coryza, is known to release outer membrane vesicles (OMVs). In t (show more...), the causative agent of infectious coryza, is known to release outer membrane vesicles (OMVs). In the present study, we investigated the composition, bioactivities, and functional properties of the OMVs of . Following extraction and purification, the OMVs were observed to be spherical in shape, with diameters ranging from 20 to 300 nm. The vesicles contained endotoxin as well as genomic DNA. The molecular weights of the OMV-contained protein fragments were mostly concentrated at 65 and 15 kDa. The components of the OMV proteins were mainly various functional enzymes (e.g., ATP-dependent RNA helicase), structural components (e.g., streptomycin B receptor and membrane protein), and some hypothetical proteins with unknown functions. The expression levels of inflammation-related factors, such as interleukin (IL)-2, IL-6, IL-1β, IL-10, and inducible nitric oxide synthase (iNOs), were significantly upregulated in chicken macrophage cells HD11 incubated with OMVs. Serum IgG antibodies were measured after two intramuscular injections of an OMV-based vaccine into specific pathogen-free (SPF) chickens. The vaccinated chickens were then challenged by of homologous and heterologous serovars. It was noted that the vaccinated chickens produced immunoglobulin G (IgG) antibodies against . The OMVs conferred an acceptable level of protection (70%), defined as an absence of colonization and of clinical signs, against the homologous strain (serovar A), while the cross-protection against heterologous challenge with serovars B and C was much weaker. However, the OMVS did provide significant protection against clinical signs for all three serovars. Overall, this study laid a foundation for further unraveling the functional roles of OMVs released by . (hide) EV-METRIC 57% (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 Cell culture supernatant Sample origin Control condition Focus vesicles Other/ Outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Density gradient Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches)/Function Sample Species Avibacterium paragallinarum Sample Type Cell culture supernatant EV-producing cells hp8 EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 235000 Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 15% Highest density fraction 45% Total gradient volume, incl. sample (mL) 14 Sample volume (mL) 2 Orientation Bottom-up Speed (g) 156000 Duration (min) 180 Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 156000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method BCA Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-300

	EV200033	1/2	Pseudomonas aeruginosa	PAO1	DG (d)(U)C UF Filtration	Dinh, Nhung Thi Hong	2020	57%
Study summary Full title Indoor dust extracellular vesicles promote cancer lung metastasis by inducing tumour necrosis factor-α All authors Nhung Thi Hong Dinh, Jaewook Lee, Jaemin Lee, Sang Soo Kim, Gyeongyun Go, Seoyoon Bae, Ye In Jun, Yae Jin Yoon, Tae-Young Roh, Yong Song Gho Journal J Extracell Vesicles Abstract Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust cont (show more...)Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust contains extracellular vesicles (EVs), which are associated with pulmonary inflammation. However, it has not been reported whether indoor dust EVs affect the cancer lung metastasis. In this study, we isolated indoor dust EVs and investigated their roles in cancer lung metastasis. Upon intranasal administration, indoor dust EVs enhanced mouse melanoma lung metastasis in a dose-dependent manner in mice. Pre-treatment or co-treatment of indoor dust EVs significantly promoted melanoma lung metastasis, whereas post-treatment of the EVs did not. In addition, the lung lysates from indoor dust EV-treated mice significantly increased tumour cell migration in vitro. We observed that tumour necrosis factor-α played important roles in indoor dust EV-mediated promotion of tumour cell migration in vitro and cancer lung metastasis in vivo. Furthermore, Pseudomonas EVs, the main components of indoor dust EVs, and indoor dust EVs showed comparable effects in promoting tumour cell migration in vitro and cancer lung metastasis in vivo. Taken together, our results suggest that indoor dust EVs, at least partly contributed by Pseudomonas EVs, are potential promoting agents of cancer lung metastasis. (hide) EV-METRIC 57% (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 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 DG (d)(U)C UF Filtration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) N/A Show all info Study aim Function Sample Species Pseudomonas aeruginosa Sample Type Cell culture supernatant EV-producing cells PAO1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 150000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 10% Highest density fraction 50% Total gradient volume, incl. sample (mL) 5.25 Sample volume (mL) 2.5 Orientation Bottom-up Rotor type SW 55 Ti Speed (g) 200000 Duration (min) 120 Fraction volume (mL) 0.5 Fraction processing None Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Polysulfone;Other Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 81.9 EM EM-type Transmission-EM Image type Wide-field

	EV190052	1/1	Solanum lycopersicum	root exudate solution	(d)(U)C Filtration	De Palma, Monica	2020	57%
Study summary Full title Plant Roots Release Small Extracellular Vesicles with Antifungal Activity All authors Monica De Palma, Alfredo Ambrosone, Antonietta Leone, Pasquale Del Gaudio, Michelina Ruocco, Lilla Turiák, Ramesh Bokka, Immacolata Fiume, Marina Tucci, Gabriella Pocsfalvi Journal Plants (Basel) Abstract Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Des (show more...)Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell and inter-kingdom communication. Despite their relevant biological implications, the existence and role of plant EVs released into the environment has been unexplored. Herein, we purified round-shaped small vesicles (EVs) by differential ultracentrifugation of a sampling solution containing root exudates of hydroponically grown tomato plants. Biophysical analyses, by means of dynamic light scattering, microfluidic resistive pulse sensing and scanning electron microscopy, showed that the size of root-released EVs range in the nanometric scale (50-100 nm). Shot-gun proteomics of tomato EVs identified 179 unique proteins, several of which are known to be involved in plant-microbe interactions. In addition, the application of root-released EVs induced a significant inhibition of spore germination and of germination tube development of the plant pathogens Fusarium oxysporum, Botrytis cinerea and Alternaria alternata. Interestingly, these EVs contain several proteins involved in plant defense, suggesting that they could be new components of the plant innate immune system. (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 root exudate solution 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 (d)(U)C Filtration Protein markers EV: non-EV: Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Solanum lycopersicum Sample Type root exudate solution Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 150000 Wash: volume per pellet (ml) 28.5 Wash: time (min) 60 Wash: Rotor Type Type 70 Ti Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method microBCA Proteomics database Yes: Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution Reported size (nm) 72 +/- 5 nm TRPS Report type Size range/distribution Reported size (nm) 51 EV concentration Yes EM EM-type Scanning electron microscopy Image type Close-up, Wide-field Report size (nm) 50-100

	EV190032	1/2	Schistosoma mansoni	NA	(d)(U)C	Marije E Kuipers	2020	57%
Study summary Full title DC-SIGN mediated internalisation of glycosylated extracellular vesicles from Schistosoma mansoni increases activation of monocyte-derived dendritic cells All authors Marije E Kuipers, Esther N M Nolte-'t Hoen, Alwin J van der Ham, Arifa Ozir-Fazalalikhan, D Linh Nguyen, Clarize M de Korne, Roman I Koning, John J Tomes, Karl F Hoffmann, Hermelijn H Smits, Cornelis H Hokke Journal J Extracell Vesicles Abstract Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host imm (show more...)Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. (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 Schisostomula (larval stage) 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 (d)(U)C Adj. k-factor 213.2 (pelleting) / 213.2 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Mechanism of uptake/transfer, Identification of content (omics approaches) Sample Species Schistosoma mansoni Sample Type Schisostomula (larval stage) culture supernatant 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: time(min) 80 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Wash: time (min) 60 Wash: Rotor Type SW 41 Ti Wash: speed (g) 120000 Wash: adjusted k-factor 213.2 Characterization: Protein analysis PMID previous EV protein analysis 26443722 Protein Concentration Method microBCA Protein Yield (µg) 6 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-650 EV concentration Yes Particle yield 23300000000 EM EM-type Transmission-EM/ Cryo-EM Image type Close-up, Wide-field Report size (nm) 35-190;30-715

	EV190032	2/2	Schistosoma mansoni	NA	(d)(U)C	Marije E Kuipers	2020	57%
Study summary Full title DC-SIGN mediated internalisation of glycosylated extracellular vesicles from Schistosoma mansoni increases activation of monocyte-derived dendritic cells All authors Marije E Kuipers, Esther N M Nolte-'t Hoen, Alwin J van der Ham, Arifa Ozir-Fazalalikhan, D Linh Nguyen, Clarize M de Korne, Roman I Koning, John J Tomes, Karl F Hoffmann, Hermelijn H Smits, Cornelis H Hokke Journal J Extracell Vesicles Abstract Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host imm (show more...)Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. (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 Schisostomula (larval stage) 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 (d)(U)C Adj. k-factor 213.2 (pelleting) / 83.21 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Mechanism of uptake/transfer, Identification of content (omics approaches) Sample Species Schistosoma mansoni Sample Type Schisostomula (larval stage) culture supernatant 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: time(min) 65 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Wash: time (min) 65 Wash: Rotor Type TLS-55 Wash: speed (g) 120000 Wash: adjusted k-factor 83.21 Characterization: Protein analysis PMID previous EV protein analysis 26443722 Protein Concentration Method microBCA Protein Yield (µg) 6 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-650 EV concentration Yes Particle yield 23300000000 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 35-190

	EV220160	1/2	Homo sapiens	MLO-Y4	(d)(U)C Filtration	Eichholz KF	2020	56%
Study summary Full title Human bone marrow stem/stromal cell osteogenesis is regulated via mechanically activated osteocyte-derived extracellular vesicles. All authors Eichholz KF, Woods I, Riffault M, Johnson GP, Corrigan M, Lowry MC, Shen N, Labour MN, Wynne K, O'Driscoll L, Hoey DA Journal Stem Cells Transl Med Abstract Bone formation or regeneration requires the recruitment, proliferation, and osteogenic differentiati (show more...)Bone formation or regeneration requires the recruitment, proliferation, and osteogenic differentiation of stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. Osteocytes are mechanosensitive cells that play fundamental roles in coordinating loading-induced bone formation via the secretion of paracrine factors. However, the exact mechanisms by which osteocytes relay mechanical signals to these progenitor cells are poorly understood. Therefore, this study aimed to demonstrate the potency of the mechanically stimulated osteocyte secretome in driving human bone marrow stem/stromal cell (hMSC) recruitment and differentiation, and characterize the secretome to identify potential factors regulating stem cell behavior and bone mechanobiology. We demonstrate that osteocytes subjected to fluid shear secrete a distinct collection of factors that significantly enhance hMSC recruitment and osteogenesis and demonstrate the key role of extracellular vesicles (EVs) in driving these effects. This demonstrates the pro-osteogenic potential of osteocyte-derived mechanically activated extracellular vesicles, which have great potential as a cell-free therapy to enhance bone regeneration and repair in diseases such as osteoporosis. (hide) EV-METRIC 56% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Statically cultured 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: TSG101/ Alix non-EV: GRP-94 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MLO-Y4 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 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) not reported Wash: time (min) 75 Wash: Rotor Type SW 32 Ti Wash: speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins TSG101/ Alix Not detected contaminants GRP-94 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 177 EM EM-type Transmission-EM Image type Wide-field

	EV200182	4/11	Mus musculus	primary oligodendrocytes	(d)(U)C DC DG	Frühbeis, Carsten	2020	56%
Study summary Full title Oligodendrocytes support axonal transport and maintenance via exosome secretion All authors Carsten Frühbeis, Wen Ping Kuo-Elsner, Christina Müller, Kerstin Barth, Leticia Peris, Stefan Tenzer, Wiebke Möbius, Hauke B Werner, Klaus-Armin Nave, Dominik Fröhlich, Eva-Maria Krämer-Albers Journal PLoS Biol Abstract Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term in (show more...)Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity. (hide) EV-METRIC 56% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin PLPko Focus vesicles exosome 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 Protein markers EV: SIRT2/ Flotillin1 non-EV: None Proteomics yes EV density (g/ml) 1.07-1.1 Show all info Study aim Function/Identification of content (omics approaches) Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells primary oligodendrocytes EV-harvesting Medium Serum free medium Cell count 3.00E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 100000 Density gradient Type Continuous Lowest density fraction 0.3M Highest density fraction 1.8M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 1 Orientation Top-down Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 60 Pelleting: rotor type TLS-55 Pelleting: speed (g) 100000 Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins Flotillin1/ SIRT2 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

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