Flow cytometry was conducted to identify cartilage stem/progenitor cells (CSPCs) using two stem cell markers CD44 and CD90. When the confluence of primary cells reached 80%, these cells were digested using EDTA-free trypsin (Gibco). After washed two times by PBS (Gibco), one million cells were resuspended in 100 μL PBS. Four experimental sets were simultaneously prepared as follows: double negative samples and single positive samples for CD44, single positive samples for CD90 and double positive samples for CD44 and CD90. Cells were incubated with 20 μL CD44 (BD pharmingen) and/or 5 μL CD90 (BD pharmingen) for 35 min on ice in darkroom. Then cells were analyzed by an Accuri C6 flow cytometer (BD pharmingen).
The apoptosis of CSPCs and chondrocytes were measured by the Annexin V/ PI apoptosis detection kit II (BD pharmingen) according to the manufacture's instruction. Briefly, one million cells were resuspended in 100 μL binding buffer after washed two times by PBS. Four experimental sets, namely double negative, single positive for annexin V and single positive for PI and double positive for annexin V and PI, were prepared by adding 5 μL PI and/or 5 μL Annexin V into binding buffer, respectively. After incubation for 30 min on ice in darkroom, the data were obtained by the Accuri C6 flow cytometer (BD pharmingen).
The apoptosis of CSPCs and chondrocytes were measured by the Annexin V/ PI apoptosis detection kit II (BD pharmingen) according to the manufacture's instruction. Briefly, one million cells were resuspended in 100 μL binding buffer after washed two times by PBS. Four experimental sets, namely double negative, single positive for annexin V and single positive for PI and double positive for annexin V and PI, were prepared by adding 5 μL PI and/or 5 μL Annexin V into binding buffer, respectively. After incubation for 30 min on ice in darkroom, the data were obtained by the Accuri C6 flow cytometer (BD pharmingen).