Anti cd31 phycoerythrin

Mouse fluorochrome-conjugated anti-human monoclonal antibodies were used. Anti-CD31 (phycoerythrin, 1:50 dilution, BD Pharmingen), anti-CD90 (fluorescein isothiocyanate, 1:50 dilution, BD Pharmingen), anti-CD45 (Vio-blue, 1:50 dilution, Miltenyi), anti-CD105 (allophycocyanin, 1:50 dilution, BD Pharmingen).
Cells were extensively washed in PBS and immunostained at 4 °C using fluochrome-coupled monoclonal antibodies diluted in FACS buffer (PBS, 1% FBS). Analyses were performed on a BD LSR-FORTESSA cytometer. Data were further analyzed using FlowJoTM software 10.1.

For the analysis of surface marker expression, ASCs were washed three times with PBS, and then incubated with a blocking solution of 3% serum in PBS for 30 minutes. After centrifugation, 1 × 10⁵ cells were suspended in blocking solution, then stained with antibodies against human CD29, CD44, CD90, CD105, and CD34 (BD Biosciences). After incubation for 30 minutes, the cells were washed with PBS, and then analyzed by flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA) following standard procedures. In order to determine cell compositions of SVF cells, multicolor flow cytometry was performed with an LSR II (BD Biosciences, San Jose, CA). The following monoclonal antibodies conjugated to fluorochromes were used: anti–CD31-phycoerythrin, anti–CD34-phycoerythrin-Cy7, and anti–CD45-fluorescein isothiocyanate (BD Biosciences, San Jose, Calif.). Cell composition percentages were calculated according to data of surface marker expression profiles.

PB mononuclear cells (PBMCs) were isolated from sodium heparinized whole blood by Ficoll-Paque density gradient centrifugation (GE Healthcare, Pittsburgh, PA, USA). Then, the phenotypes of lymphocytes were determined using flow cytometry. Briefly, PBMCs were stained with the following fluorochrome conjugated monoclonal antibodies: anti-CD3-peridin chlorophyll protein (Percp), anti-CD4-Percp, anti-CD8-Percp, anti-CD31-phycoerythrin (PE), anti-CXCR4-allophycocyanin (APC), and isotype-matched control IgG antibodies (all from BD Biosciences, San Diego, CA, USA) for 30 min at room temperature, according to the manufacturer's instructions. After being washed with PBS, a minimum of 20,000 events per tube was acquired using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using CellQuest software (BD Biosciences) and FlowJo 7.6.1 software (Tree Star).