Fitc conjugated antibody against c kit

For blood sampling at different time points (baseline, and at 18 h or on days 14 after induction of CLI), blood sampling from tail vein was performed using a 25# needle. After treatment with red blood cell-lysing buffer, the cells remained were labeled with appropriate antibodies. Flow cytometric analysis for identification of cell surface markers was performed based on our previous reports [50 (link)]. Briefly, the cells were incubated for 30 min with primary antibodies, including PE-conjugated antibodies (against CD34, Sca-1, CD31, BD Biosciences, San Jose, CA, USA), FITC-conjugated antibody against c-Kit (BD Biosciences), anti-CXCR4 (Abcam, Cambridge, MA, USA), and anti-KDR (NeoMarkers, Fremont, CA, USA) antibodies which were further recognized by Alexa flour 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Isotype-identical antibodies (IgG) served as controls. Flow cytometric analyses were performed by utilizing a fluorescence-activated cell sorter (Beckman Coulter FC500 flow cytometer, Brea, CA, USA).

The procedure and protocol of flow cytometric analysis was according to our recent reports [7 (link), 8 (link)]. In detail, peripheral blood sampling at baseline and at 18 h and on day 14 after CLI induction was obtained via cardiac puncture with a 30# needle. Moreover, the BM levels of EPCs were measured at 18 h and on day 14 after CLI induction by needle aspiration and washing from the long bone of the animals. After treatment with red blood cell-lysing buffer, the cells were labeled with appropriate antibodies. Flow cytometric quantification of EPCs through identification of the chosen cell surface markers was performed based on our recent reports [7 (link), 8 (link)]. Briefly, the cells were incubated for 30 minutes with primary antibodies, including PE-conjugated antibodies (against Sca-1 and CD31, BD Biosciences), FITC-conjugated antibody against c-Kit (BD Biosciences), and anti-KDR (NeoMarkers) antibodies which were further recognized by Alexa flour 488-conjugated secondary antibodies (Invitrogen). Isotype-identical antibodies (IgG) served as controls. Flow cytometric analyses were performed by utilizing a fluorescence-activated cell sorter (Beckman Coulter FC500 flow cytometer).