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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210214	1/1	Homo sapiens	primary neutrophils	(d)(U)C UF SEC (non-commercial)	Bonifay, Amandine	2022	57%
Study summary Full title A new strategy to count and sort neutrophil‐derived extracellular vesicles: Validation in infectious disorders All authors Amandine Bonifay, Stéphane Robert, Belinda Champagne, Paul‐Rémi Petit, Aude Eugène, Corinne Chareyre, Anne‐Claire Duchez, Mélanie Vélier, Shirley Fritz, Loris Vallier, Romaric Lacroix, Françoise Dignat‐George Journal J Extracell Vesicles Abstract Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcel (show more...)Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles large extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Ultrafiltration Size-exclusion chromatography (non-commercial) Protein markers EV: CD63/ GAPDH/ Integrin-beta3-subunit/ CD15/ CD66b/ CD44/ CD11c non-EV: Albumin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells primary neutrophils EV-harvesting Medium Serum-containing medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Ultra filtration Cut-off size (kDa) 100 Membrane type Polyethersulfone (PES) Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 0.5 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ GAPDH/ Integrin-beta3-subunit Detected contaminants Albumin Flow cytometry Type of Flow cytometry Cytoflex S Calibration bead size 0.1 0.16 0.22 0.24 0.3 0.5 0.9 Antibody details provided? Yes Detected EV-associated proteins CD15/ CD66b Not detected EV-associated proteins CD44/ CD11c Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Median Reported size (nm) 191 Particle analysis: flow cytometry Flow cytometer type Cytoflex S Hardware adjustment Calibration bead size 0.1 0.16 0.22 0.24 0.3 0.5 0.9 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

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EV-TRACK ID	EV210214
species	Homo sapiens
sample type	Cell culture
cell type	primary neutrophils
condition	Control condition
separation protocol	dUC/ Ultrafiltration/ Size-exclusion chromatography (non-commercial)
Exp. nr.	1
EV-METRIC %	57