Flow cytometry analysis

Chondrocyte apoptosis was evaluated using an Annexin FITC/PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions following transfection and treatment. Chondrocytes were harvested, washed with PBS (Beijing Solarbio Science & Technology Co., Ltd.), resuspended at a concentration of 1.0x10⁶ cells/ml and incubated with 5 µl Annexin V-FITC and 10 µl PI for 10 min at room temperature in the dark. Chondrocyte apoptotic rate was analyzed using a FACScan^® flow cytometer (BD Biosciences) for 1 h. The level of apoptotic cells were analyzed using FlowJo 7.6.1. (FlowJo LLC). Apoptosis rate was calculated as the sum of the early apoptosis rate (the lower right quadrant) and the late apoptosis rate (the upper right quadrant).