Propidium Iodide (PI) is a red fluorescent dye utilized for cell staining and is suitable for fluorescence microscopy, confocal microscopy, flow cytometry, and fluorometer analysis. In aqueous solution, the Ex/Em of PI is 493/636 nm. Upon binding with nucleic acid, the Ex/Em shifts to 535/617 nm, enhancing the fluorescence signal 20-30 times.

METHODS: Propidium Iodide uptake and Flow cytometry to detect cell death:
1. Store the PI stock solution (0.5 mg/mL in PBS) in a dark place at 4℃. Immediately before use, prepare PI-FACS buffer by adding 20 µL of PI stock solution per 1 mL of PBS.
2. Collect suspended cells: Collect cells directly in centrifuge tubes and centrifuge at 500g for 5 min to harvest all cells.
3. Collect adherent cells: Remove and preserve the medium containing dead and mitotic cells. Isolate live cells using standard tissue culture techniques, such as incubation with trypsin-EDTA, and be sure to collect any washings (e.g., PBS). Add cells from the culture medium and cells from any wash solution to the isolated cells and centrifuge at 500 g for 5 min to harvest all cells.
4. Resuspend the harvested cells in PI-FACS buffer. The cells were incubated in the dark for 15 min at room temperature.
5. Determine the cell mortality rate by flow cytometry. [1]