Characterization of CDC Surface Markers

The expression levels of c-kit, CD34, CD90 and CD105 in CDCs were estimated by immunostaining17 (link). In brief, twice-passaged CDCs (1 × 10⁴/well) were cultured in 8-well chamber culture slides (Lab-Tek, Thermo Scientific Nunc) coated with 15 μg/ml fibronectin. The cells were fixed in 4% paraformaldehyde for 10 minutes after 3 days of culture. After blocking, the cells were incubated with PE-conjugated rat anti-mouse c-kit antibody (eBioscience), FITC-conjugated rat anti-mouse CD34 antibody (eBioscience), or with rat anti-mouse CD90 monoclonal antibody (Abcam) and rat anti-mouse CD105 monoclonal antibody (Abcam) followed by donkey anti-rat Alexa Flour 488-conjugated secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylin-dole (DAPI), and the positively stained cells were counted under a fluorescent microscope with 200-fold magnification. Twenty fields per slide were randomly selected for quantitative counting.