Cd34 pe

Fourth passage ASCs were cultured in complete media until sub-confluent prior to flow cytometric analysis (Becton Dickinson FACSCanto cytometer, Franklin Lakes, NJ). Media was removed by washing the ASCs with PBS. The ASCs were then harvested with 0.25% trypsin/EDTA. Cell viability was assessed via propidium iodide staining. Determination of fluorescent cells, dead cells, debris, and background noise was made with FACS DIVA software (Becton Dickinson, Franklin Lakes, NJ). For extracellular staining, the cells were treated with a mixture of ice-cold 1% sodium azide for 30 min, washed with PBS, and incubated with 3% bovine serum albumin (BSA) for 30 min on ice. Following a PBS wash, aliquots of ASCs were incubated in BSA with the following monoclonal antibodies for flow cytometry: CD73-Alexa 488 (BD Biosciences, San Jose, CA), CD90-Alexa 488 (BioLegend, San Diego, CA), CD11b-Alexa 488 (BioLegend, San Diego, CA), CD34-PE (Santa Cruz Biotechnology, Dallas, TX), CD36-Alexa 488 (BioLegend, San Diego, CA), and CD29-PECy7 (BioLegend, San Diego, CA). For intracellular staining, cells were counted and fixed with 4% paraformaldehyde for 20 min at room temperature, washed three times with PBS, and lysed with 90% ice-cold methanol for 30 min. The lysed cells were incubated in 3% BSA for 30 min and treated with a monoclonal primary antibody to CD105-PE (Bioss Inc., Woburn, MA).

Cells at passage three were digested, washed with PBS and incubated with antibodies against rat CD31-PE (BD Biosciences, New Jersey, USA), CD34-PE (Santa Cruz, Dallas, USA), CD45-FITC (BD Biosciences, New Jersey, USA), and CD90-PE (BD Biosciences, New Jersey, USA), as well as with isotype IgG1-PE (BD Biosciences, New Jersey, USA) and isotype IgG1-FITC (BD Biosciences, New Jersey, USA) for 30 min. Cells were then washed and centrifuged at 1500 rpm for 5 min. Finally, cells (5×10⁵/ml) were suspended in PBS and analyzed by flow cytometry.