Anti cd146

Fresh blood samples were stained for anti-CD45 (to exclude hematopoietic cells) (eBioscience, San Diego, CA), anti-CD31 (an endothelial marker) (BD Biosciences, San Jose, CA), anti-CD133 (a progenitor cell marker) (MiltenyiBiotec, Auburn, CA), anti-CD146 (an endothelial cell marker) (Millipore, Temecula, CA) and the apoptosis marker 7-aminoactinomycin D (7AAD) (Merck, Buenos Aires, S.A.). The red blood cells were lysed by red cell lysis buffer and CECs and CEPs were analyzed by FACS (BD Biosciences). CECs were defined as negative for CD45, positive for CD146 and CD31, and negative for the progenitor marker CD133. CEPs were depicted by expression of CD133.

nDPSC were harvested by trypsin/EDTA treatment for 5 min at 37 °C. After inactivation of trypsin and centrifugation (5 min at 300 g), 100 µl of the cell suspension (10⁵ cells) was incubated for 30 min at room temperature in the dark with the following fluorochrome-conjugated antibodies: anti-CD10 (eBiosciences; Clone: CB-CALLA), anti-CD13 (eBiosciences; Clone: WM-15), anti-CD29 (Chemicon, Clone: TDM29), anti-CD34 (Biolegend; Clone: 4H11), anti-CD44 (Caltag; Clone: MEM-85), anti-CD45 (Caltag; Clone: HI30), anti-CD71 (Biolegend; Clone: MEM-75), anti-CD73 (BD Pharming; Clone: AD2), anti-CD90 (Chemicon; Clone: F15-42-1), anti-CD105 (Caltag; Clone: SN6), anti-CD146 (Millipore; Clone: P1H12), anti-CD166 (Beckman; Clone: 3A6), anti-CD222 (eBiosciences; Clone: MEM-238), anti-CD271 (Biolegend; Clone: ME20.4), anti-CXCR4 (Caltag; Clone: 12G5), anti-INF-beta (INFsource; Clone: MMHB-3) and anti-HLA-DR (Caltag; Clone: Tu36). After washing with PBS, at least 10,000 events were acquired using a Beckman Coulter FC500 flow cytometer. Instrument settings and gating strategies were established using isotype controls, and data were analysed using Kaluza software (Beckman Coulter). All experiments were carried out in duplicates.

The frozen samples were thawed just prior to analysis. MPs with procoagulant potential were measured using a solid-phase capture assay from a commercial kit (Zymuphen MP-activity kit; Hyphen BioMed, France). In brief, MPs were isolated by capture onto immobilized annexin V, and the amount of captured MPs was determined by a prothrombinase assay using their procoagulant potential. The solid-phase capture assay combined with the prothrombinase assay provides a functional assessment of the procoagulant potential of isolated circulating MPs, regardless of the capture ligand [12 (link)]. Based on the median value of MPs, we divided our participants into either a low MPs group (The cell origins of the MPs were determined by antigenic capture with insolubilized specific antibodies instead of annexin V using similar solid-phase capture methods [12 (link)]. In the present study, the following biotinylated monoclonal antibodies were used: anti-CD31, anti-CD42b (Abcam, Cambridge, UK), and anti-CD146 (Millipore, Billerica, MA, USA). Results were expressed as phosphatidylserine equivalents (PS eq), which were calculated using the standard calibration curve constructed using liposomes of known concentration. All tests were performed in duplicate.