Anti human cd31

Human PDL cells were grown in two T25 dishes (Greiner Bio One, Monroe, NC, USA) to confluency. The cells were then trypsinized and resuspended in flow cytometry buffer (eBioscience, San Diego, CA, USA). These cells were subsequently blocked for non-specific interactions using human Fc receptor binding inhibitor (eBioscience, San Diego, CA, USA) at room temperature. For surface staining we used the following antibodies: rat mAb CD44-FITC (1:50 dilution, ab19622, Abcam, Cambridge, MA, USA), anti-CD146 (1:40 dilution, 50–1469, eBioscience, San Diego, CA, USA) and anti-human CD31 (1:40 dilution, 11–0319, eBioscience, San Diego, CA, USA), and incubated for 30 min in the dark at 2–8°C. The cells were washed three times with flow cytometry buffer after centrifugation at 400–600xg for 5 min. After the final wash, the cells were suspended in flow cytometry buffer and 7-AAD staining (eBioscience, San Diego, CA, USA), before being fixed with intracellular fixation buffer (eBioscience, San Diego, CA, USA). The tubes were then analyzed using a LSRII cytometer equipped with 488 nm, 561 nm, 640 nm, 405 nm and 350 nm lasers and all flow cytometry data were analyzed with Flow Jo software. Side and forward scatter of aggregates in cell lysates were determined using log scale plots. Voltage settings for the PerCp-A, FITC and efluoro660 channels were kept constant for all experiments described.