Anti mouse fitc conjugated secondary antibody

Chondrocytes (1×10⁶⁾ from growth plate (passage 1) were washed in PBS and incubated with corresponding primary antibodies for one hour at 4°C. Primary antibodies used here were conjugated antibodies specific for CD44-FITC (dilution 1:100, #550974; BD Biosciences, San Diego, CA, USA), CD29-PE (dilution 1:80, #48-0291; eBiosciences; Thermo Fisher Scientific, Inc.), CD34-PE (dilution 1:100; #sc-74499 PE; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), CD45-PE (dilution 1:80, #12-0461; eBiosciences; Thermo Fisher Scientific, Inc.) and unconjugated antibodies for CD105 (dilution 1:100, #ab11414) and CD146 (dilution 1:80, #ab75769) (both from Abcam, Cambridge, MA, USA). After washing with PBS, cells stained with CD105 and CD146 were incubated with anti-mouse FITC-conjugated secondary antibody (dilution 1:500, #A0568; Beyotime Institute of Biotechnology, Guangzhou, China) or Alexa Fluor 594 donkey anti-rabbit IgG (dilution 1:500, #A-21207; Invitrogen; Life Technologies) for 30 min at 4°C. The cells were washed twice and then re-suspended in 200 µl PBS for the flow cytometry analysis (FACSort; BD Biosciences).

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).