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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200112	1/2	Homo sapiens	HUVEC	(d)(U)C	Giraud, Romain	2022	67%
Study summary Full title Tracking Radiolabeled Endothelial Microvesicles Predicts Their Therapeutic Efficacy: A Proof-of-Concept Study in Peripheral Ischemia Mouse Model Using SPECT/CT Imaging All authors Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue Journal Pharmaceutics Abstract Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (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 (Differential) (ultra)centrifugation No extra separation steps Protein markers EV: CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1/ integrin beta 3 non-EV: Albumin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 70000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type JA-30.50 Wash: speed (g) 70000 Other Name other separation method No extra separation steps Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD51/ caveolin/ integrin-beta3/ CD81 Not detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD146/ ICAM1/ CD31 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 434.5 EM EM-type Transmission-EM Image type Close-up Report size (nm) 434

	EV200112	2/2	Homo sapiens	HUVEC	(d)(U)C qEV	Giraud, Romain	2022	67%
Study summary Full title Tracking Radiolabeled Endothelial Microvesicles Predicts Their Therapeutic Efficacy: A Proof-of-Concept Study in Peripheral Ischemia Mouse Model Using SPECT/CT Imaging All authors Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue Journal Pharmaceutics Abstract Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (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 (Differential) (ultra)centrifugation Commercial method Protein markers EV: beta 3 integrin/ CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1 non-EV: Albumin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HUVEC 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 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type JA-30.50 Pelleting: speed (g) 70000 Wash: volume per pellet (ml) 30 Wash: time (min) 90 Wash: Rotor Type JA-30.50 Wash: speed (g) 70000 Commercial kit qEV Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ CD51/ caveolin/ integrin-beta3/ CD81 Not detected contaminants Albumin Flow cytometry aspecific beads Antibody details provided? No Detected EV-associated proteins CD31/ CD146/ ICAM1 Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 434.5 EM EM-type Transmission-EM Image type Close-up Report size (nm) 407

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EV-TRACK ID	EV200112
species	Homo sapiens
sample type	Cell culture
cell type	HUVEC
condition	Control condition
separation protocol	dUC No extra separation steps	dUC qEV
Exp. nr.	1	2
EV-METRIC %	67	67