Flow cytometer instrument

Chondrocytes were seeded at a density of 2 × 10⁶ cells/well in 6-well plates and cultured for 24 h, then treated with SNP (0.5 mM) and 2-APB (100 μM) for 12 h. ROS in cells was measured by DCFH-DA fluorescence intensity. The original medium on the culture plate was replaced with DMEM without FBS. After addition of DCFH-DA (1:1,000) (Beyotime, China), the culture plate was incubated for 20 min in the incubator at 37°C and washed three times with DMEM without FBS, and images obtained under a fluorescence microscope. ROS expression was assessed via flow cytometry. FBS-free DMEM was used for dilution of DCFH-DA (1:1000). The chondrocytes were digested with trypsin, and the cell suspension was centrifuged at 1500 rpm for 5 min at 4°C. Then the collected chondrocytes were suspended in diluted DCFH-DA, placed in a cell incubator at 37°C for 20 min. The cell suspension was collected by centrifugation at 4°C and 1500 rpm for 5 min, and then washed three times in DMEM without FBS. Chondrocytes were measured on a flow cytometer instrument (Beckman, United Ststes).

For measurement of apoptosis, cells were seeded at a density of 2 × 10⁶ cells/well in 6-well plates and cultured for 24 h, then treated with SNP (0.5 mM) and 2-APB (100 μM) for 12 h. Collected cells were washed and re-suspended in binding buffer. Annexin V-FITC (5 μl) was added to samples and incubated for 15 min in the dark, followed by addition of 10 μl PI for 5 min in the dark. Apoptosis was measured by a flow cytometer instrument (Beckman, United States).