Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used to stain cells. When PI is bound to nucleic acids, the fluorescence excitation maximum is 535 nm and the emission maximum is 617 nm. Excitation energy can be supplied with a xenon or mercury-arc lamp or with the 488 line of an argon-ion laser. Propidium iodide is used as a DNA stain for both flow cytometry, to evaluate cell viability or DNA content in cell cycle analysis, and microscopy to visualise the nucleus and other DNA-containing organelles. It can be used to differentiate necrotic, apoptotic and normal cells.
Hi Ahmad,
Propidium iodide is a vital stain. a live cells exclude the dye and cannot be stained. It stains only dead cells.
It is possible depending on the definition of the word "viable", which can be equivocal.
If it is viable in the sense of PI-staining, then of course not; but it may just mean that the cell has "just" a somewhat compromised plasmic membrane.
If it is in the sense of ongoing apoptosis, necrosis, other cell death mechanisms, or loss of efflux pump activity, then you may consider probing the specific process that causes the corresponding loss of viability.
It will also be dependant on the concentration of PI that you are using.
Hope this helps, and good luck!
Gary Leslie Wright, Thank for your answer. Can you please elaborate to some extent? What type of transporter carries PI inside the cells, while not being compromised cell membrane. What is the conc. of PI (and volume) required to stain 200µl of cell suspension? I will highly appreciate your response. Many thanks.
I don't think there is a special transporter to transport PI inside.But Gary is right, "over staining" is possible.
We use a 0.2 mg / mL PI solution. From this solution we add 2 µL for 200µL cell suspension (incubate 10 minutes at RT). So final PI concentration will be 2 µg / mL. Works good for flow cytometry.
It is function of concentration of PI and and staining time. Find lowest/shortest concentration/incubation time of PI that stains your dead cells (kill some for positive) and stick with it. I only mentioned transporters because cancer cells expression some odd ones. Use organic transporter blockers if you suspect that PI entry is mediated by a translocase.
If you need to introduce some molecules inside a living cell, you can try to use liposomes. When PI is inside the cytoplasm, it can stain also mRNAs.
Hi all, I am facing a similar situation in which my untreated cells stain negative for PI, while treated cells stain positive. These treated cells look quite happy under the microscope, though, so I am not convinced that viabilty is reduced so massively. Could someone please suggest an assay to check whether the plasma membrane is compromised? Thank you.
Hi Mona, Which cells or cell lines you are using? Which drugs you are treating the cells with? Is it a lipopeptide (e.g., polymyxins)?
Hi, I am using neuroblastoma cells and performing a knockdown experiment for a transcriptional CDK.
Did you try a cross control by the neutral red assay? (see the protocol :Neutral red uptake assay for the estimation of cell viability/cytotoxicity, by
Guillermo Repetto, Ana del Peso & Jorge L Zurita
Published online 12 June 2008; )
An early stage of dying cells is the lost of plasma membrane integrity that in turn a small molecule likes PI can passivly diffuse and binds to DNA. Incubation time with PI and a buffer that cells are suspended play a crucial role. During incubation with PI, ice cold 0.1% BSA in 1X PBS solution will protect the plasma membrane. Try this it could help.
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