Annexin 5 fitc propidium iodide apoptosis kit

The cell cycle kit and Annexin V-FITC/propidium iodide (PI) apoptosis kit (4A Biotech, Beijing, China) were used according to the manufacturer’s instructions. H1975 cells were harvested and fixed in 1 ml 75% ethanol for 24 h. The supernatant was discarded, and the cells were re-suspended in 0.5 ml cold PBS containing 10 μg/ml RNase and 20 μg/ml PI stock solution. Subsequently, they were transferred to FACS tubes and incubated in the dark for 30 min at RT. Cell cycle was assayed by flow cytometry (BD, Biosciences) at 488 nm. For apoptosis assay, adherent cells were harvested after 24 h of treatment and re-suspended in 100 μl binding buffer (1 × 10⁶ cells/ml), then incubated with 5 μl Annexin V–FITC for 5 min in the dark at RT. A 10 μl PI (20 μg/ml) and 400 μl binding buffer were added to the cells, which were then immediately analyzed by flow cytometry at 630 and 525 nm.

For apoptosis analysis, an Annexin V-FITC/propidium iodide (PI) apoptosis kit (4 A Biotech, Beijing, China) was used under the manufacturer’s instructions. Control or PRN1 knockdown MCF7 cells were harvested, washed with PBS, and re-suspended in 100 µL binding buffer (2 × 10⁵ cells). After an incubation with 5 µL Annexin V–FITC for 5 min at room temperature in dark, 5 µL PI and 400 µL binding buffer were added to the cells for a 20-min incubation at room temperature in dark. The stained cells were examined by flow cytometer as instructed by its manufacturer. The data were analyzed with a flowjo software. Annexin V positive indicates that cells are in early apoptosis, while both FITC Annexin V and PI positive in late apoptosis or already dead [20 (link), 21 (link)].