Basic questions about PI staining in Flowcyto - (Sep/06/2010 )

- Is there a time limit to run samples after addition of PI stain? because I ran my samples 2 hours after addition of PI, and healthy cells and apoptotic cells show same results. maybe PI stains healthy nuclei if kept for long?

- what buffer do you dissolve your PI in? I dissolve in PBS and just add 300ul to my fixed cell pellet before running experiments

- I am getting areas for my unstained cells on the diagrams. So there is no need to stain healthy cells, is it? because the machine reads them anyway!

- why do we need to add RNAse?

I am not sure if there is a time limit but I like to look at my cells right after staining. I am not very keen to leave cell suspension in stain for too long.
PI I use is dissolved in PBS, have to check percentage, on top of the head 1 %. And following staining cells need to be washed really well, PI gives lots of background.
Cells always have some autoflorescence so ideally stained healthy cells will be included as a control.
Oh, samples need to be RNase treated because PI binds to RNA, producing false positives.

kajmak on Sat Sep 11 23:30:09 2010 said:

I am a bit confused about the purpose of your PI staining. Are you using it for cell cycle analysis or for life/dead staining?
If for cell cycle analysis, then you should certainly wash your cells.
If for life/dead staining, then you do not need to add RNase A.

?

rsm

I'm checking the cells after transfection with a gfp-tagged viral protein. The protein kills the cells and I want to measure the percentage of dead cells.

- I do not fix cells that are expressing GFP because when I fix the intensity drops.

- I also found that PI staining of fixed GFP-expressing cells wipes the GFP areas. so now I do not fix or stain those.

but I haven't tried if PI staining of un-fixed cells is effective ?! maybe dead cells have broken membranes and PI can get in easily without any need to fix cells. This way live cells won't take up the stain. Because after fixation almost all cells take up PI. dead or live doesn't matter.

I have not used RNase so far but I get lots of background near the zero values on dot blot. don't know what they are

Some recommendations... Don't use PI, that really interferes with GFP. You'll have all your GFP positive cells also positive for PI. Better use ToPro3, at 1:10000 dilution. It shows in the APC channel.

If you want to use PI, make some controls: GFP only (compensate for PI signal), PI only (same for GFP), unstained, both stained. Use PI at 50ng/ml final concentration (add 1:1000 directly into your cell suspension, no wash needed). Your living cells are negative for PI. No need to fix, otherwise all cells are dead = positive for PI.

Anyway, I think you will underestimate the number of dead cells. For the preparation of cells before FACS, you'll wash and trypsinize and wash again etc. So you will lose all dead cells during washes. Better think about a different approach... Or you should measure apoptosis in GFP positive cells.

Cheers,

rsm

Thanks so much rsm,

I noticed that PI interferes with GFP, that's why I don't stain GFP expressing cells.

I also use a lot of controls, stained and unstained, normal or dead cells.

However I don't think I lose any dead cells during washings. cells which are blebbing and are undergoing apoptosis still have enough weight to get pulled down by centrifugation. Besides I always save the medium with floating cells and add the trypsinized cells to those and centrifuge all together. I some times also scrape the cells if I want to run the experiment urgently, though I don't like it because it will break many cells.

Is the only purpose of gfp fusion viral protein localization or something else?
If so I would do it differently. Do protein localization (gfp fusion) with constitutive promoter and viral protein only (for cel death) alone without gfp with inducible promoter. Or just seperately, viral protein + gfp and viral protein, whatever method you are using now. That way you are sure that gfp is not interfering with protein function and PI staining can be done afterwards.

If protein is from animal or plant virus make sure you try the same thing in animal or plant cells. I tried one gene in yeast and it killed it. When I tried it in plant cells it helped them survive.

There are some other stains you could try to confirm apoptosis in yeast.