Pe mouse anti human cd31

The present study used UC-MSCs in third passages, which had been collected and cultured according to the requirements of the Ethics Committee of the Department of Biotherapy Center of the Third Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). The UC-MSCs were cultured in a humidified 37°C, 5% CO₂ incubator with Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.). Surface markers of the UC-MSCs were confirmed by staining with the following antibodies: PE mouse anti-human CD31, FITC mouse anti-human CD34, PE mouse anti-human CD44, FITC mouse anti-human CD45, PE mouse anti-human CD73, FITC mouse anti-human CD90, and PE mouse anti-human CD105 (BD Biosciences, New Jersey, USA). Suitable isotype controls were also used. The UC-MSCs were stained with each antibody for 30 min at 4°C and analyzed with a flow cytometer (FACS Verse TM, BD Biosciences, USA) and Flow Jo 7.6 (Treestar, Ashland, Oregon, USA). The UC-MSCs in the third passage were used in this study [10 ].

Cells were pretreated with Accutase (Invitrogen) for 3 minutes at 37°C and then washed with cold PBS. After 300×g centrifugation, 2×10⁵ cells were resuspended in 100 ml of PBS and incubated with primary antibodies for 30 minutes at 4°C. Then, the cells were washed by using PBS with 2% FBS and 0.2 mM EDTA and used for flow cytometry analysis or sorting. The antibodies were as follows: Human APJ-APC-conjugated Antibody (R&D), PE Mouse Anti-Human CD31 (BD Biosciences), APC Mouse Anti-Human CD34 (BD Biosciences), APC Mouse Anti-Human CD43 (BD Biosciences), Mouse IgG3 APC-conjugated Antibody (R&D), PE Mouse IgG1, κ Isotype Control (BD Biosciences).