Apc anti cd19 clone sj25c1

EV samples were analyzed on FACS CANTO II using DIVA software (Becton Dickinson, BD Biosciences, San Jose, CA, USA) as previously reported (18 (link)). Briefly, EV samples were labeled with 40 µM of 5-carboxyfluorescein diacetate-succinimidil ester (CFDA-SE, Sigma-Aldrich) and then were incubated with 100 ng of different conjugated antibodies for specific surface markers and their respective controls for 40 min at 37°C in a reaction volume of 30 µL. Specifically, phycoerythrin (PE) anti-CD14 (clone M5E2; BD), allophycocyanin (APC) anti-CD19 (clone SJ25C1; BD), PE anti-CD33 (clone P67.6; BD), APC anti-CD34 (clone 8G12; BD), APC anti-CD38 (clone HB-7; BD) and PE anti-CD117 (clone 104D2; BD) were used. After labeling, 3 µL of EV samples were transferred in TruCount tubes (BD) containing 400 µL of 0.02 µm filtered PBS. A total of 50,000 events were immediately acquired in low flow rate.
EV concentration (EVs/mL) was calculated with the formula EVs/mL=G_EV/G_TC*TC/V* DF (G_EV= number of events CFSE⁺ in specific antibody positive EV gate, G_TC=number of events in TruCount bead gate, TC= number of TruCount beads in single TruCount tube, V= sample volume used in the analysis, DF= sample dilution factor).