Apc conjugated anti cd31

Two million to 5 million cells per condition were incubated in 250 µL of 2% FBS/PBS with fluorochrome-conjugated primary antibodies for 30 min with shaking every 10 min. Primary antibodies were washed with 2% FBS/PBS, and cells were resuspended in 2.5 mg/mL DAPI (Invitrogen) before analysis. The following primary antibodies were used for the analysis of K14rtTA/TetO-Cre/Rosa-YFP mice: PECy7-conjugated anti-CD24 (1:100; BD Biosciences, clone M1/69), APC-conjugated anti-CD29 (1:100; eBiosciences, clone eBioHMb1-1), PE-conjugated anti-CD45 (1:100; eBiosciences, clone 30-F11), PE-conjugated anti-CD31 (1:100; BD Biosciences, clone MEC 13.33), and PE-conjugated anti-CD140a (1:100; eBiosciences, clone APA5). Data analysis was performed on a BD LSR Fortessa using FACS DiVa software (BD Biosciences). Dead cells were excluded with DAPI; CD45⁺, CD31⁺, and CD140a⁺ cells were excluded (Lin⁺) before analysis of the YFP⁺ cells. Primary antibodies used for the analysis of K8rtTA/TetO-Cre/Rosa-tdTomato mice were PECy7-conjugated anti-CD24 (1:100; BD Biosciences, clone M1/69), FITC-conjugated anti-CD29 (1:100; BD Biosciences, clone Ha2/5), APC-conjugated anti-CD45 (1:100; eBiosciences, clone 30-F11), APC-conjugated anti-CD31 (1:100; eBiosciences, clone 390), and APC-conjugated anti-CD140a (1:100; eBiosciences, clone APA5). A minimum of five 10⁵ total events per mouse were analyzed.

HUVEC (Promocell, c-12203), were cultured in endothelial cell growth medium (PromoCell; c-22010), supplemented with 35 µg/mL gentamycin and endothelial cell growth supplements (Promocell, c-39215) at 5% CO₂ and 37 °C. HUVEC were routinely tested for mycoplasma contamination. HUVEC were seeded into 24-well plates (at 1.5 × 10⁵/well) 24 h prior to drug treatment. HUVEC were washed to remove cell debris and constitutively released EVs then pre-treated for 30 min in endothelial cell growth media with the appropriate inhibitor. After this, media was replaced with HEPES-buffered saline supplemented with glucose (0.9 mg/ml), CaCl₂ (2 mM), A23187 (10 μM) and inhibitor for 10 min. Following stimulation, both the EV-rich medium and trypsinized endothelial cells were collected and stained with APC-conjugated anti-CD31 (eBioscience,ThermoFisher, UK). Samples were analysed using a BD Accuri C6 flow cytometer. APC fluorescence (FL4) was used to trigger event acquisition and identify endothelial (or endothelial-derived EV) events. to determine EV count and cellular PS exposure, respectively. PS-exposing endothelial-derived EVs were defined as CD31⁺/annexin V⁺ events that were smaller than 1 µm in an analogous manner to platelet-derived EVs.