Novoexpress 1

The change in cell size was evaluated using a NovoCyte flow cytometer (ACEA Biosciences, Hangzhou, China). Forward scatter (FSC) is proportional to the size of the cell. Larger cells have stronger FSC signals. Side scatter (SSC) is related to cell granularity and structural complexity. Cells were treated with CoCl₂ or 2 mM NAC for 24 hours and then digested and centrifuged for 3 minutes at 1000 rpm. After resuspension in DMEM, 3 × 10⁴ cells per sample were tested, and cell debris was excluded by gating. The values of forward scatter height (FSC-H) and side scatter height (SSC-H) were measured and analyzed with NovoExpress 1.2.5 software (ACEA Biosciences, Hangzhou, China).
A cellular ROS kit was used to evaluate ROS levels induced by hypoxia. Briefly, cells were handled following the manufacturer's instructions. The green fluorescence of DCF was detected using the FITC channel at 488 nm excitation and 530 nm emission. For the apoptosis assay, a FITC Annexin V Apoptosis Kit (BD, NJ, USA) was used. Briefly, the cells were handled following the manufacturer's instructions. Green (Annexin) and red (PI) fluorescence was detected using the FITC and PE channels, respectively. The data were analyzed with NovoExpress 1.2.5 software.

Cell cycle analysis kit (Beyotime, Shanghai, China) was adopted to check cell cycle distribution of samples through Propidium Iodide (PI) staining. Cells were collected by centrifugation at 310 × g for 5 min followed by fixation with ice-cold 70% ethanol for 2 h at 4°C. The cells were resuspended with 500 μl staining buffer solution (Beyotime) and then incubated with 25 μl PI (Beyotime) and 10 μl RNase A solution (Beyotime) in dark for 30 min at 37°C. Annexin V-FITC apoptosis detection kit (Beyotime) was utilized to check cell apoptosis of samples by double-staining of Annexin V-FITC and PI. Cells were collected by centrifugation at 1000 × g for 5 min followed by resuspension with 195 µl Annexin V-FITC binding solution (Beyotime). Then, the cells were incubated with 5 μl AnnexinV-FITC and 10 μl PI at room temperature for 15 min. Finally, all cell samples were detected using NovoCyte Flow Cytometers (Acea Biosciences, San Diego, CA, USA) and analyzed using the NovoExpress 1.2.5 software (ACEA Bioscience). The apoptosis rate of cells was calculated as follows: apoptosis rate = (early apoptosis (Annexin V⁺ and PI⁻) cells + late apoptosis (Annexin V⁺ and PI⁺) cells)/total cells × 100%.