c-Kit/CD117

Kalam K. Chan ; Danna M. Breen ; Jiwanjeet K. Dhaliwall ; Michael R. Ward ; Nael Al Koudsi ; Loretta Lam ; Melissa De Souza ; Husam Ghanim ; Paresh Dandona ; Duncan Stewart ; Michelle P. Bendeck ; and Adria Giacca. Insulin Increases Reendothelialization and Inhibits Cell Migration and Neointimal Growth After Arterial Injury. Arteriosclerosis, Thrombosis, and Vascular Biology. 2009. doi: 10.1161/ATVBAHA.109.185447.

Flow Cytometry. Blood was obtained at baseline and 3 and 7 days after carotid injury in separate rats by cardiac puncture and drawn into Sodium Citrate (9:1 v/v) Vacutainer tubes (BD Biosciences). For each staining sample, 200 μl of blood was mixed with 1.0 mL RBC lysis buffer (eBioscience) and incubated for 10 min at room temperature in the dark. Tubes were centrifuged and the supernatant discarded. The remaining cells (pellet) were suspended in 100 μl of PBE staining buffer, and appropriate primary antibody (mouse anti-rat VEGFR2 (Novus Biologicals), rabbit anti-rat c-kit (Neuromics), and rabbit anti-rat sca-1(Novus Biologicals) was incubated with the cells at room temperature in the dark for 30 minutes. Following washing (2x 1.0 ml PBE buffer) and centrifugation, cells were exposed to secondary antibody (FITC donkey anti-rabbit IgG) or Straptavidin-APC for 30 minutes in the dark. Following two additional wash and centrifugations steps, cells were suspended in PBS. The number of gated cells positive for the three markers was quantified by flow cytometry (Beckman Coulter Cytomics FC500) using FL1 (FITC for c-kit and sca-1) and FL4 (for VEGFR2) filters.