Propidium iodide is a small, aromatic compound that is commonly used in research and development as a fluorescent nucleic acid binding stain. PI is often used to differentiate between apoptotic and necrotic cells from healthy cells as it freely penetrates cell membranes of dead or dying cells but is excluded from viable cells. Propidium iodide stains nucleic acids, like DNA and RNA, and can therefore be used as an indicator of membrane integrity in flow cytometry, in situ hybridization, and immunohistochemistry applications.

After addition of propidium iodide to a sample, cells with permanently or reversibly damaged cell membranes will fluoresce red. In this way, propidium iodide makes it possible to identify and quantitate dead and necrotic cells rapidly and reliably. Propidium iodide helps determine overall cell viability, which is an important parameter used to monitor the response of a cell population to cytotoxic drugs or other environmental factors.

Viability studies typically include a propidium iodide stain, coupled with the apoptosis marker stain (such as Annexin V-FITC or iFluor® 488 that has a bright green fluorescence). When used together, total cell counts can be obtained and the morphologies of cells within a sample may be observed. For example, intact cells (iFluor® 488-PI-), early apoptotic (iFluor® 488+PI-) and late apoptotic or necrotic cells (iFluor® 488+PI+) can all be discriminated by using these two stains in tandem.

A basic protocol for staining cells with PI is described below:

This protocol should be adapted based on experimental requirements and optimization.  

The major advantage of using propidium iodide staining techniques is that these methods cause minimal cell loss and cell damage by hydrolysis can be avoided. There are, however, a few common issues with propidium iodide. Conventional Annexin V/PI protocols, as well as similar staining protocols, may lead to a significant number of false positive events (>40%). Such false positives are associated with how propidium iodide will also stain RNA within the cytoplasmic compartment. This issue can occur in both primary cells and cell lines and occurs most often with larger cells that have a smaller nuclear-to-cytoplasmic-ratio.

Since propidium iodide is a membrane impermeable DNA-binding stain, it is generally also used alongside a membrane-permeable DNA-binding counterstain in bacterial viability studies. However, in the research, propidium iodide staining of adherent cells in biofilms has shown to significantly underestimate the actual bacterial viability due to the presence of extracellular nucleic acids (eNA). Upon observation, it appears that a false dead layer of red, propidium iodide-stained cells will appear. This occurrence can sometimes cover up a subpopulation of double-stained cells that have green interiors, indicating viability, under a red layer which hints at DNA being stained outside of intact membranes.

Therefore, viability staining results of such cell populations should always be validated by an alternative method for estimating viability, e.g., confocal laser scanning microscopy (CLSM) or fluorescence microscopy. The use of propidium iodide, in most cases, is limited to fixed or permeabilized cells to allow the dye to enter into the cells that would be otherwise actively pumped out of a living cell. In permeabilization and fixation, alcohol or aldehydes are usually choice reagents. Importantly, aldehyde and alcohol are often incompatible with fluorescent proteins and some surface markers, so paraformaldehyde may be a more appropriate solution. As far as safety goes, propidium iodide is a suspected carcinogen, and may cause skin, eye, and respiratory irritation.