It should be OK to add PI directly to the treated cells, unless you are testing fluorescent compounds that give a fluorescent signal at the same wavelength as PI. However, sometimes we remove part of the medium before imaging to obtain a more clear signal, but usually the final results in both cases were very similar. Just be careful with the concentration and incubation time of cells with PI - after longer period and at higher concentrations we noticed its penetration also to the live cells.

I Agree with Vilma, it can be observed that way but if your concentration is high or you take too long it will start entering live cells. What you have to do if you can´t wash is to stain the well or plate and look it at the microscope, then stain the next and look it at the microscope and so on. Maybe you can stain more than one well but you have be careful not to take more than a few minutes.

Hope it helps!

Thank you so much Vilma and Diego!. Very helpful information. May I ask what concentration and incubation time you have used before? Also I read that RNAse treatment of the sample is necessary as PI binds to RNA as well and gives around 40% false positive results. What are your thoughts on it?

I believe RNAse is required when you are analyzing it with flow citometry, witch is a lot more sensitive, I never used it when analyzing with fluorescence. The incubation time we used was 2 minutes, however, I don´t remember the concentration. Propidium iodide is very toxic and it is dangerous to weight it duo to inhalation risk, be aware of that. If the drug has not been open yet you can dilute the whole drug with appropriate volume.

Hope it helps!

Thank you so much Diego for your thoughtful insights.

I agree with what's been said, but a couple of additional notes: RNase treatment is not necessary for live/dead (since "dead" is indicated by any incorporation into cells, whether binding to DNA or RNA). Rather, RNase is more for getting a clean nuclear (DNA) signal or for (more commonly) use for cell cycle analysis (where labeling of only DNA is crucial).

I also wanted to add that PI will have a very low background signal in the media. Usually it's not a problem, but if you find the background is too high, then dropping the concentration is what you'd want to do. You might also want to use a non-autofluorescent buffer, such as FluoroBrite DMEM.

Oh, and if your drug compound is present during imaging, be aware that some compounds autofluoresce, leading to background. You might want to test this with a no-dye control.

Thank you Jason for your thoughtful reply.

Propidium iodide is useful for adherent cells.

Hi all, I agree with Jason, I did not have a problem with false positive cells yet even after 72h incubation during time lapse imaging. Might depend on cell type.

We observed that it might take more time for PI to stain the DNA of a necrotic cell than for a cytosolic stain (in our case YFP) to get out when the plasma membrane is disrupted. So you might want to allow for 20-30min incubation with PI.

Hi and thanks to all for the useful information about PI staining. Has any of you have a protocol to stain live/dead adherent cells with propidium iodide and analyze it by imaging? I have check some articles and people usually combine the PI with Hoescht or DAPI, does it make any sense?

We did it that way. You first stain 1 or 2 minutes with IP, washed two times, then fix cells ans stain 5 or 10 minutes (i don remember well) with dapi.

Hi Lucas

you can work with live cells all along if your imaging device supports live cells (stage incubation chamber). In that case stain 1ug/ml Hoechst and same concentration PI for at least 1h (takes a while for the live cells to take up Hoechst). You can leave the staining in there while imaging because it basically only shows up when bound to DNA. You can also stain in medium but if it contains Phenol Red you might get a quite high background.

If you cannot work with live cells use the same concentrations and protocol but fix the cells afterwards. If you need to permeabilise and immune stain as well you might loose the PI staining...

Best

Heiko

Hi all, anyone has the experience staining the adherent cells with both PI and cytoplasmic protein immunostaining ? since the cell permeabilization would give the false positive result of PI staining, isnt it ?

any suggestion would be very appreciated..

best regards