Apoptosis analysis kit

Flow cytometry and Annexin V-FITC/PI were used to examine CEP cell apoptosis. An Apoptosis Analysis Kit (AO2001-02P-H, Sungene, Biotech) was used to stain CEP cells according to the manufacturer’s instructions. Briefly, CEP cells were incubated with 5 µL of Annexin V-FITC for 10 min in the dark after being resuspended. Then, the CEP cells were incubated with 5 µL of PI for 5 min in the dark. Finally, apoptosis was measured by flow cytometry (CytoFLEX, Beckman Coulter, USA).
DNA damage in CEP cells was evaluated by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) staining. CEP cells were fixed and stained with a TUNEL Cell Apoptosis Detection Kit (G1504, Servicebio Biological) according to the manufacturer’s instructions, and the nuclei were stained with DAPI.

The indicated number of cells were seeded in 24-well plates, cultured, and treated with various compounds for 24h. An apoptosis analysis kit (Tianjin Sungene Biotech Co., Ltd.) was applied to detect the apoptosis status of cells by flow cytometry (LSR II, BD Biosciences). Cell differentiation was measured by the fluorescence intensity of CD11b. Cells were resuspended in PI/RNase staining buffer for further cell cycle analysis. For proliferation analysis, cells were cocultured (with SD49-7 or an equivalent volume of DMSO) for 72h and were counted every 24h by the trypan blue exclusion cell counting method.

For apoptosis analysis, cells were washed twice with 1× binding buffer then labelled with Annexin V and propidium iodide (PI) following the manufacturer’s instructions. The Apoptosis Analysis Kit was ordered from Tianjin Sungene Biotech (Tianjing, China). For cell cycle analysis, cells were washed twice with ice-cold PBS and fixed with 70% ethanol at −20 °C overnight. Then, the cells were washed with PBS and resuspended with a solution containing 50 μg/ml PI with 100 μg/ml RNase A in the dark at 37 °C for 30 min. The analysis of the samples was performed by flow cytometry (Becton-Dickinson, San Jose, CA, USA), and the acquired data were analysed with the CellQuest software (BD Biosciences).