Cd31 pe

For flow cytometric analysis, cells were dissociated into single cell suspensions by incubation in trypsin/EDTA for 5 min. The dissociated cells were resuspended (1 × 10⁵ cells) in 100uL of FACS buffer (PBS + 5% FBS + 0.1% Sodium azide), and then incubated for 30 minutes at 4 °C. After washing twice with FACS buffer, cells were analyzed with a fluorescence-activated cell sorter (FACS) (FACS Canto II, Becton Dickinson, San Jose, CA), and the acquired data were analyzed (Cell Quest software, Becton Dickinson, San Jose, CA). Cultured ASCs were characterized using antibodies against rat CD73 (BD Biosciences, 551123), CD90-FITC (BD Biosciences, 554897), CD31-PE (BD Biosciences, 555027), CD34 (Santa Cruz Biotechnology, sc-7324), and CD45-FITC (BD Biosciences, 554877). Anti-CD34 and CD73 antibodies were labeled with Alexa Fluor 488 (Thermo Fisher Scientific) Species-specific fluorophore (Alexa-Fluor 488) conjugated secondary antibodies (Abcam).
Lentiviral vector-transfected RLMVECs were analyzed by FACS with mouse anti-rat CD90-FITC and CD31-PE to determine whether they retained their vascular phenotype (Supplementary Fig. 5).

The International Society for Cell Therapy previously suggested the following minimal criteria to define human MSCs [18] (link): expression of CD105, CD73, and CD90, and no expression of CD45, CD34, CD14, CD11b, CD79α, CD19, or HLA-DR surface molecules. Cells were harvested by treatment with 0.05% trypsin-EDTA (Sigma-Aldrich, St Louis, MO) for 3 min at 37°C, recovered by centrifugation at 400× g for 5 min, washed twice in ice-cold PBS containing 2% FBS, and re-suspended at 1×10⁵ cells/antibody test. The expression of specific MSC markers was assessed using the following antibodies: CD14-FITC, CD31-PE, CD105-FITC, CD34-PE, CD45-PE, CD73-PE, and CD90-APC (all purchased from Abcam, Cambridge, UK). Negative control staining was performed using FITC/PE/APC-conjugated mouse IgG₁ isotype antibodies (Abcam). After incubation for 30 min at room temperature in the dark, the cells were washed with PBS, resuspended in 100 µL PBS, and analyzed by a MACSQuant flow cytometer (Miltenyi Biotec, Gladbach, Germany).