Propidium Iodide Stain

1. Add Hoechst 33342 to the cell sample media at 0.5% v/v. For example, add 1.5 µL Hoechst to 300 µL of cells. 2. Incubate 10-20 minutes at room temperature., 3. Visualize with a fluorescence microscope by using a UV excitation and blue emission filter. The blue Hoechst stain fluoresces at 461 nm., 4. Alternatively, cells may be analyzed with a flow cytometer using a UV excitation source., 5. When bound to dsDNA, the maximum absorption is 350 nm and the maximum emission is 461.

Propidium iodide (PI, Catalog 638) is an intercalating red fluorescent reagent that binds between the base pairs of DNA in membrane-compromised cells and is used to identify necrotic and apoptotic cells. As PI is membrane impermeant, it cannot reach the DNA in viable cells, thus allowing the identification of cells with permeabilized membranes in a population. PI distinguishes between living and dead cells by counterstaining nucleic acids red in necrotic, dead, apoptotic and membrane-compromised cells, while the DNA in healthy cells remains unstained. Propidium iodide can be used with ICT’s FAM-FLICA® caspase inhibitor reagents (such as Catalog 92) to identify four populations of cells: living; early apoptotic; late apoptotic; and necrotic (Figures 2 and 3). Propidium iodide is provided ready-to-use at 250 µg/mL. Just add it to the cell culture media, incubate for a few minutes, and analyze. One molecule of PI stoichiometrically binds every four to five base pairs of DNA. Unbound PI excites at 488-492 nm and exhibits an emission maximum at 635 nm. Upon binding to DNA, the fluorescence of PI is enhanced 20-30 fold. When bound to nucleic acids, the maximum absorption is 535 nm and the maximum emission is 617 nm (Figure 1). Cells may be viewed through a fluorescence microscope or analyzed with a flow cytometer. PI is for research use only. Not for use in diagnostic procedures.