Pi staining solution

ARPE-19 cells were digested with 0.25% trypsin (Beyotime, Haimen, Jiangsu, China) and centrifuged at 1000 rpm for 5 min at 4 °C. The cells were then harvested. The trypsinization time should not be too long to prevent false positives. The cells were washed twice with pre-cooled PBS, centrifuged at 1000 rpm for 5 min at 4 °C, and 1–5 × 10⁵ cells were collected. Next, the PBS was discarded and 100 μl of 1 × binding buffer was added to resuspend the cells. The cells were mixed gently with 5 μL of V-FITC and 10 μL of PI staining solution (BestBio, Shanghai, China). The cells were incubated with the dyes for 10–15 min at room temperature in the dark. After the incubation, 400 μL of 1 × binding buffer was added to the cells, mixed and placed on ice. After 1 hour, the sample was detected by flow cytometry. The V-FITC-positive and PI-negative cells were recorded as apoptotic cells, and the percentage of apoptotic cells in each group was recorded.

For apoptosis assay, organoids were first digested into single-cell suspensions and then centrifuged at 800 g for 5 min. Then cells were suspended with 400 μL binding buffer containing 5 μL of Annexin V-FITC and 10 μL of PI staining solution (BestBio, BB-4101). After staining for 5 min, flow cytometry was used to distinguish dead and apoptotic cell populations. As for the cell cycle analysis, approximately 400,000 cells per well were first implanted into 6-well plates for 12 h and then drugs were added. Next, FxCycle™ PI/RNase Staining Solution (Life Technologies, F10797) was used to perform cell cycle analysis. At least three replications were performed for each experiment.