Mouse anti cd105 antibody

To confirm the expression of CD105 and CD146 in sorted cells, immunofluorescence staining was performed. Cells were grown to sub confluence on coverslips in 12-wells plate. Cells were then fixed with 4% paraformaldehyde for 20 min at room temperature and washed 3 times with PBS. After that, the cells were incubated with 0.1% Triton X-100 for 15 min at room temperature (RT) and blocked with 1% bovine serum albumin (Biosharp, Hefei, China) for 1 h at room temperature. Then the cells were incubated with mouse anti-CD105 antibody (dilution 1:100) or rabbit anti-CD146 antibody (dilution 1:80) (both from Abcam) overnight at 4°C. After washing with PBS, cells stained with CD105 or CD146 were incubated with anti-mouse FITC-conjugated secondary antibody (dilution 1:500, #A0568; Beyotime Institute of Biotechnology) or Alexa Fluor 594 donkey anti-rabbit IgG (dilusion 1:1,000, #A-21207; Invitrogen; Life Technologies) for 1 h at room temperature. The nuclei were then labeled with DAPI (50 µg/ml; Sigma-Aldrich; Merck KGaA) for 5 min. Fluorescence images were captured by fluorescence microscopy (FV500; Olympus Corporation).

About 5×10⁶ growth plate chondrocytes were harvested for magnetic activated cell sorting (Miltenyi, Teterow, Germany) according to manufacturer's instructions. Briefly, single-cell suspensions were incubated with mouse anti-CD105 antibody (dilution 1:100) or rabbit anti-CD146 antibody (dilution 1:80) (both from Abcam) for 30 min at 37°C. After centrifugated at 300 × g for 10 min and washed by PBS twice, the cells were then incubated with anti-rabbit IgG microbeads or anti-mouse IgG microbeads for 15 min at 37°C. The cells were then washed and resuspended with washing buffer and added into the MS column, the CD146⁺ SSCs and CD105⁺ SSCs were obtained following the instructions. The sorted cells were cultured in growth medium to obtain sufficient quantities for further research.