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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170008	1/3	Homo sapiens	primary monocyte derived dendritic cell	DG (d)(U)C	Mercedes Tkach	2017	100%
Study summary Full title Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. All authors Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C Journal EMBO J Abstract Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Protein markers EV: CD63/ MHC2/ CD9/ CD9,MHC2 non-EV: None Proteomics no Show all info Study aim EV functional activity Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary monocyte derived dendritic cell EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g 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) 20 Pelleting: rotor type Swinging bucket Pelleting: speed (g) 2000 Wash: time (min) 20 Wash: Rotor Type Eppendorf 5810R cf; swinging bucket Wash: speed (g) 2000 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 1.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2.5 Pelleting: duration (min) 30 Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 65.69 EV-subtype Distinction between multiple subtypes Centrifugation steps: 2K, 10K, 100K PMID previous EV protein analysis 26858453 Protein Concentration Method microBCA Protein Yield (µg) 2.9+-0.3 Western Blot Detected EV-associated proteins CD9,MHC2 Flow cytometry Type of Flow cytometry MACSQuant Miltenyi Calibration bead size 0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis 26858453 Extra particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield 6.52E10+-2.03E10 particles/million cells EM EM-type Transmission-EM/ Scanning-EM EM protein MHC2 Image type Close-up, Wide-field

	EV170008	2/3	Homo sapiens	primary monocyte derived dendritic cell	DG (d)(U)C	Mercedes Tkach	2017	100%
Study summary Full title Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. All authors Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C Journal EMBO J Abstract Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 2097 (pelleting) / 2097 (washing) Protein markers EV: CD63/ MHC2/ CD9/ CD9,MHC2 non-EV: None Proteomics no Show all info Study aim EV functional activity Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary monocyte derived dendritic cell EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 40 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 2097. Wash: time (min) 40 Wash: Rotor Type Type 45 Ti Wash: speed (g) 10000 Wash: adjusted k-factor 2097. Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 1.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2.5 Pelleting: duration (min) 30 Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 65.69 EV-subtype Distinction between multiple subtypes Centrifugation steps: 2K, 10K, 100K PMID previous EV protein analysis 26858453 Protein Concentration Method microBCA Protein Yield (µg) 1.7+-0.3 Western Blot Detected EV-associated proteins CD9,MHC2 Flow cytometry Type of Flow cytometry MACSQuant Miltenyi Calibration bead size 0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis 26858453 Extra particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield 4.95E10+-3.81E10 particles/million cells EM EM-type Transmission-EM/ Scanning-EM EM protein MHC2 Image type Close-up, Wide-field

	EV170008	3/3	Homo sapiens	primary monocyte derived dendritic cell	DG (d)(U)C	Mercedes Tkach	2017	100%
Study summary Full title Qualitative differences in T-cell activation by dendritic cell-derived extracellular vesicle subtypes. All authors Tkach M, Kowal J, Zucchetti AE, Enserink L, Jouve M, Lankar D, Saitakis M, Martin-Jaular L, Théry C Journal EMBO J Abstract Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnosti (show more...)Exosomes, nano-sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4+ T-cell activation in vitro When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN-γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN-γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Adj. k-factor 209.7 (pelleting) / 209.7 (washing) Protein markers EV: CD63/ MHC2/ CD9/ CD9,MHC2 non-EV: None Proteomics no Show all info Study aim EV functional activity Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary monocyte derived dendritic cell EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA 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) 90 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 209.7 Wash: time (min) 90 Wash: Rotor Type Type 45 Ti Wash: speed (g) 100000 Wash: adjusted k-factor 209.7 Density gradient Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0.1 Highest density fraction 0.3 Sample volume (mL) 1.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 55 Ti Speed (g) 350000 Duration (min) 60 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 2.5 Pelleting: duration (min) 30 Pelleting: rotor type TLA-110 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 65.69 EV-subtype Distinction between multiple subtypes Centrifugation steps: 2K, 10K, 100K PMID previous EV protein analysis 26858453 Protein Concentration Method microBCA Protein Yield (µg) 1.1+-0.2 Western Blot Detected EV-associated proteins CD9,MHC2 Flow cytometry Type of Flow cytometry MACSQuant Miltenyi Calibration bead size 0.1-0.3,0.4-0.6,0.7-0.9,1.0-1.9 Characterization: Lipid analysis No Characterization: Particle analysis PMID previous EV particle analysis 26858453 Extra particle analysis NTA Report type Size range/distribution EV concentration Yes Particle yield 3.76E10+-1.41E10 particles/million cells EM EM-type Transmission-EM/ Scanning-EM EM protein MHC2 Image type Close-up, Wide-field

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EV-TRACK ID	EV170008
species	Homo sapiens
sample type	Cell culture
cell type	primary monocyte derived dendritic cell
condition	Control condition
separation protocol	DG (d)(U)C	DG (d)(U)C	DG (d)(U)C
Exp. nr.	1	2	3
EV-METRIC %	100	100	100