Dapi staining solution

SMSCs were seeded on glass coverslips in 24-well plates and treated as the experiment group processing. After another 24 hours, the chondrogenic induction cells were fixed and subsequently treated with 0.3% Triton X-100 (MP Biomedicals, USA). After washed with PBS, the cells were then incubated in 5% BSA. Then, the cells were incubated with microtubule-associated protein light chain 3 (LC3) rabbit monoclonal antibody (1 : 100; Abcam, USA) overnight at 4°C. The negative control group was treated with 1% BSA instead of the primary antibody. The FITC-conjugated goat antirabbit IgG (1 : 100; Proteintech, USA) was added for 1 hour and then incubated with DAPI staining solution (Leagene, China) for 15 min. The coverslip was dried and sealed with antifluorescence quenching agent (Beyotime, China). The cells were observed under laser confocal microscopy (FV10i, Olympus, Japan).

EA.hy926 cells were cultured on coverslips, treated with bolus addition of H₂O₂ or/and 7, 8-DHF for 24 hrs and stained with DAPI staining solution (Leagene Biotech, Beijing, China). The stained nuclei were observed under Leica DMI 4000B fluorescent microscope (Leica Microsystems, Wetzlar, Germany).
The treated EA.hy926 cells were digested with trypsin without EDTA added, collected and suspended in a binding buffer at a cell density of 1x10⁶/mL. Cells were mixed with an Annexin V-FITC/PI Apoptosis Detection Kit (Invitrogen/Life Technologies, Carlsbad, CA, USA) as instructed by the manufacturer, filtered with 200 mesh screens and passed through the flow cytometer(BD FACSCalibur, BD Biosciences, San Jose, USA) for apoptotic detection and analyses.