Fitc conjugated rat anti mouse cd45

Phase-contrast microscopy was used to evaluate the morphology of CPCs, and flow cytometric analysis was performed to examine the expression of stem cell surface markers. CPCs were trypsinised, re-suspended in phosphate-buffered saline (PBS) and blocked with 3% FBS for 15 minutes for flow cytometric analysis. Next, CPCs were labelled with FITC-conjugated rat anti-mouse c-kit (BD Biosciences, USA), FITC-conjugated rat anti-mouse Scal-1 (BD Biosciences, USA), FITC-conjugated rat anti-mouse CD34 (MiltenyiBiotec Inc., GER) and FITC-conjugated rat anti-mouse CD45 (MiltenyiBiotec Inc., GER) at 4 °C in a dark room for 30 minutes and then washed twice with cold PBS. Mouse IgG1 antibody (BD Biosciences, USA) was used as an isotype control. Data were collected from 1 × 10⁵ cells using a FACS Calibur flow cytometer (BD Biosciences, USA) and analysed using WinMDI software.

C-kit-positive cells were isolated from the expanded cells using MACS, as described above. We performed a flow cytometry analysis to analyze the stem cell phenotypes. CSCs were isolated by enzymatic digestion using accutase, re-suspended in phosphate-buffered saline (PBS) and blocked with 3% fetal bovine serum for 15 min prior to the flow cytometry analysis. A nonspecific mouse IgG1 antibody (BD Biosciences, USA) was used as a control. Subsequently, CSCs were labeled with FITC-conjugated rat anti-mouse c-kit, FITC-conjugated rat anti-mouse spinocerebellar ataxia type 1 (Sca-1; BD Biosciences, USA), FITC-conjugated rat anti-mouse CD34 (Miltenyi Biotec Inc., Germany) and FITC-conjugated rat anti-mouse CD45 (Miltenyi Biotec Inc., Germany) antibodies for 30 min at 4 °C in the dark, washed twice with cold PBS, and then re-suspended in buffer. The data were obtained with a FACSCalibur flow cytometer (BD Biosciences, USA) and analyzed using WinMDI software.