Modifit software

After being cultivated in 12-well plates with 1 × 10⁵ cellular structures/well JEG-3 and JAR cellular structures per well for 24 hours in serum-free RPMI-1640 media, the cellular structures were shaken for 5 min at 150 g at a speed of g. then twice rinsed with precooled 1PBS. The cellular structures were then preserved in 70% ethanol (CEF 590056, Solarbio, Peking, China) for 15 minutes at 20 degrees, centrifugation at 150 g for 5 minutes, and washed twice with chilled 1PBS. To eliminate residual RNA, cellular structures were treated for 45 minutes at 37 degrees with 10 g/L DNase-free RNase A (Sigma, Saint Louis, US). After two washes with ice-cold 1PBS, the cellular structures were centrifuged at 150 g for 5 minutes and then treated with 1 mg/mL iodide (Sigma, Saint Louis, US) for 12 minutes at 4°C in the dark. This action was taken to remove any residual RNA. Olympus, Tokyo, Japan, used FACS analysis to evaluate the distribution of cellular structures at each phase of the cell cycle and to identify cell apoptosis using an Annexin-FITC/PI apoptosis Identification Kit (Beyotime, Nanjing, China). All FACS data were processed and analyzed with ModiFit software (Olympus, Tokyo, Japan).

A total of 1 × 10⁵ chondrocytes were cultured in a 12-well plate using serum-free DMEM for 24 h, centrifuged at 1500 g for 5 min and washed with pre-cooled 1 × PBS twice. Subsequently, cells were fixed with 70% ethanol (Solarbio, Beijing, China), placed at 20 °C for 15 min, then centrifuged at 1500 g for 5 min, and washed twice with pre-chilled 1 × PBS. Subsequently, cells were incubated with 10 μg/μL DNase-free RNaseA (Sigma, St. Louis, USA) for 45 min at 37 °C to eliminate RNA, and washed twice with pre-chilled 1 × PBS. Finally, following centrifuging at 150g for 5 min, the cells were incubated with 1 mg/mL iodide (Sigma, St. Louis, USA) in the dark at 4 °C for 12 min. The percentage of cells at each cell cycle phase is quantified in a flow cytometer and analyzed by ModiFit software (Olympus, Tokyo, Japan). Besides, according to instructions, Annexin-FITC/PI Apoptosis Detection Kit (Beyotime, Nanjing, China) was combined with flow cytometry to detect cell apoptosis, according to manufacturer’s instructions [17 (link)]. In addition, modified software (Olympus, Tokyo, Japan) was performed to analyze the data.