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Propidium Iodide

Propidium Iodide (PI) is a stain that greatly increases fluorescence upon DNA intercalation. PI has an almost identical excitation and emission spectrum to EthidiumBromide (EtBr) and is efficiently excited by the 488 nm argon laser line in flow cytometry. PI emission is detected with approximately equivalent intensity in either of the traditional orange or red fluorescent cytometry detectors. Isolated nuclei (see: NusseNuclearPreparation) and cells that have been permeablized either by ethanol or paraformaldehyde can have their DNA quantified for cell cycle analysis by PI staining. PI can also distinguish dead cells from live cells since a live cell will have an intact cytoplasmic membrane that excludes PI and that will therefor be non-fluorescent. PI is a superior choice than ethidium bromide for live/dead staining because propidium is more highly charged causing more complete exclusion from live cells -- ethidium is capable of low-level passage through intact cell membranes. For cell cycle analysis, PI and EtBr may be used interchangeably. Loss of outer membrane integrity allowing PI staining of DNA is a fairly late-stage indicator of cell death. Earlier stages of cell death can be monitored by Annexin-5 detection of membrane-everted phosphatidylserine instead.

Note: Dead cells increase their fluorescence in the presence of PI approximately 500x, so it is advisable to use a 4-decade log scale for cytometric discrimination of live from dead cells.

stock solution:
1 mg/ml propidium iodide in water
store at 4C in the dark

typical working final concentration:
2 μg/ml in buffered solution such as PBS

Caution: Propidium iodide is (like ethidium) a strong mutagen. Be very careful when making a stock solution from the dried powder to avoid spreading/inhaling fine propidium iodide dust.

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