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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230045	1/4	Homo sapiens	hMSC-TERT	(d)(U)C Filtration	L Ramos T	2016	56%
Study summary Full title MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry. All authors L Ramos T, Sánchez-Abarca LI, Muntión S, Preciado S, Puig N, López-Ruano G, Hernández-Hernández Á, Redondo A, Ortega R, Rodríguez C, Sánchez-Guijo F, del Cañizo C Journal Cell Commun Signal Abstract Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodula (show more...)Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ CD9/ CD73 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hMSC-TERT 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 70ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) not reported Wash: time (min) 70 Wash: Rotor Type 70ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ CD73 Flow cytometry Type of Flow cytometry FACSCanto II (BD) Calibration bead size 0.5/ 0.9/ 3 Antibody details provided? Yes Detected EV-associated proteins CD9/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 159.7 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230045	2/4	Homo sapiens	hMSC-GFP	(d)(U)C Filtration	L Ramos T	2016	56%
Study summary Full title MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry. All authors L Ramos T, Sánchez-Abarca LI, Muntión S, Preciado S, Puig N, López-Ruano G, Hernández-Hernández Á, Redondo A, Ortega R, Rodríguez C, Sánchez-Guijo F, del Cañizo C Journal Cell Commun Signal Abstract Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodula (show more...)Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ CD73 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hMSC-GFP 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 70ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) not reported Wash: time (min) 70 Wash: Rotor Type 70ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ CD73 Flow cytometry Type of Flow cytometry FACSCanto II (BD) Calibration bead size 0.5/ 0.9/ 3 Antibody details provided? Yes Detected EV-associated proteins CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 136.6 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230045	3/4	Homo sapiens	HS-5	(d)(U)C Filtration	L Ramos T	2016	56%
Study summary Full title MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry. All authors L Ramos T, Sánchez-Abarca LI, Muntión S, Preciado S, Puig N, López-Ruano G, Hernández-Hernández Á, Redondo A, Ortega R, Rodríguez C, Sánchez-Guijo F, del Cañizo C Journal Cell Commun Signal Abstract Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodula (show more...)Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ CD73 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HS-5 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 70ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) not reported Wash: time (min) 70 Wash: Rotor Type 70ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ CD73 Flow cytometry Type of Flow cytometry FACSCanto II (BD) Calibration bead size 0.5/ 0.9/ 3 Antibody details provided? Yes Detected EV-associated proteins CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 125.6 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV230045	4/4	Homo sapiens	K562	(d)(U)C Filtration	L Ramos T	2016	56%
Study summary Full title MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry. All authors L Ramos T, Sánchez-Abarca LI, Muntión S, Preciado S, Puig N, López-Ruano G, Hernández-Hernández Á, Redondo A, Ortega R, Rodríguez C, Sánchez-Guijo F, del Cañizo C Journal Cell Commun Signal Abstract Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodula (show more...)Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ CD73 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells K562 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 70ti Pelleting: speed (g) 100000 Wash: volume per pellet (ml) not reported Wash: time (min) 70 Wash: Rotor Type 70ti Wash: speed (g) 100000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD81/ CD73 Flow cytometry Type of Flow cytometry FACSCanto II (BD) Calibration bead size 0.5/ 0.9/ 3 Antibody details provided? Yes Detected EV-associated proteins CD63/ CD81 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 122 EV concentration Yes EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV230045
species	Homo sapiens
sample type	Cell culture
cell type	hMSC-TERT	hMSC-GFP	HS-5	K562
condition	Control condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2	3	4
EV-METRIC %	56	56	56	56