Annexin V-FITC/PI Apoptosis Assay Protocol

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)].