Anti cd106 fitc

After incubations, the PLTs were removed from cultures; HUVECs were washed once with PBS and detached with trypsin/EDTA solution trypsin (0.25%). Antibodies used for flow cytometry to recognize endothelial adhesion molecules were all purchased from BD Pharmingen (San Diego, CA, USA); anti-CD62P-fluorescein isothiocyanate (FITC; P-selectin; clone AK-4), PAC-1-FITC (recognizes α_IIbβ₃ in its activated conformation), anti-CD54-phycoerythin (PE; ICAM-1, clone LB-2), anti-CD106-FITC (VCAM-1, clone 5110C9) and anti-CD62E-Allophycocyanin (APC; E-selectin, clone 68-5H11). Flow cytometric analysis (10 000 events) was performed using a FACSCalibur flow cytometer and BD FACS DIVA 7.0 software (BD, San Jose, CA, USA). Possible alterations in HUVEC phenotype and activation state following trypsin treatment should not be overlooked; however control and experimental HUVEC were treated with trypsin in a similar manner and differences between groups were therefore not the consequence of trypsin application.

Expression of EMMPRIN and adhesion molecules on EVs was performed by flow cytometry using anti-CD63-coated microbeads following the manufacturer’s protocol (Exosome-Human CD63 Isolation/Detection Reagent, Invitrogen). The following fluorochrome-conjugated antibodies were tested: anti-CD56-FITC, anti-CD49b-FITC, anti-CD106-FITC and anti-CD197-FITC (all from BD Pharmingen Franklin Lakes, NJ, USA); anti-ICAM1-PE and anti-ICAM2-PE (both from Biolegend, San Diego, CA, USA); anti-alphaVbeta3-FITC and anti-CD147-FITC (both from eBioscienceTM). The binding of a specific antibody was evaluated using the ratio between the MFI of beads-bound EVs incubated with isotype-matched non-reactive fluorochrome-conjugated antibodies and the MFI of the beads-bound EVs incubated with each specific antibody. Ratios greater than 1 indicate expression of the protein. Analysis was performed using a Navios EX flow cytometer (Beckman Coulter) with software Navios (Beckman Coulter).