Anti cd73 apc

Expression of cell surface molecules of canine AT-MSCs at passage 3 was evaluated by flow cytometry. Briefly, cells were harvested from cell flasks by enzymatic digestion with 0.25% trypsin-EDTA (Sigma-Aldrich). Next, cells were stained with conjugated antibodies (anti-CD45-Alexa 647, anti-CD19-FITC, anti-CD14-PE, anti-CD73-APC, anti-CD90-APC, anti-CD105-PE, and anti-CD44-FITC; all from eBioscience, San Diego, CA, USA) diluted in PBS containing 0.5% BSA. AT-MSCs were incubated for 20 min in the dark at 4°C. Following incubation, cells were washed with PBS and centrifuged at 300 ×g for 5 min. Cell pellets were resuspended with PBS for data acquisition using a BD FACSAria™ II flow cytometer. Data were analyzed using FlowJo software (FlowJo LCC, Ashland, OR, USA).

BM-MSCs of passages 3–5 or transfected BM-MSCs were harvested and incubated with anti-CD90-FITC (11-0909), anti-CD90-PE (12-0909), anti-CD105-PE (12-1057), anti-CD73-APC (17-0739), anti-CD45-FITC (11-9459), anti-CD45-PE (12-9459), anti-CD34-PE (12-0349), anti-CD19-APC (17-0199), and anti-CD11b-PE (12-0113, eBioscience) antibodies at 4°C for 30 minutes. Results were detected by using an FC 500 MCL Flow Cytometer (Beckman Coulter). Appropriate isotype-matched antibodies (eBioscience) were applied as controls.