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.

HEK293 cells:1.1 μM

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]

Propidium Iodide absorption and flow cytometry assay for cell death
1. Store PI stock solution (0.5 mg/mL in PBS) at 4°C in the dark. Immediately before use, prepare PI-FACS buffer by adding 20 µL of PI stock solution to every 1 mL of PBS.
2. Collect suspended cells: Collect cells directly in a centrifuge tube and harvest all cells by centrifugation at 500g for 5 min.
3. Collect adherent cells: Remove and save the culture 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 wash solution (such as PBS). Add cells in the culture medium and cells from any wash solution to the isolated cells and harvest all cells by centrifugation at 500g for 5 min.
4. Resuspend the harvested cells in PI-FACS buffer. Incubate the cells in the dark at room temperature for 15 min.
5. Determine cell death rate by flow cytometry.