I'm quite new in flow cytometry, especially the PI staining; what I did to see dead cells was following: after incubation with my antibodies and final spindown, I resuspended the cells in PBS and then added PI just before measuring. After doing this, I looked at the populations in forward/side scatter dot plot, then selected each of them, visualizing the degree of PI-staining of each.
What I see everytime on PI histogram is that I get 4 populations, two being on the left, other two showing a shift to the right - and a small portion of these pairs intersect... According to the the typical textbook knowledge the two pairs being on the left should be unstained, therefore living, while the other two are probably living. BUT, looking back at the scatter it seems that the living population is the one having the smallest size with least complexity (what one would say are dead ones)!!
Isn't this controversial, or am I just reading the PI histogram wrong? I attached an image depicting my problem for better understanding, which populations should I take??
By the way, I'm using HEKT cells to transfect two proteins with different extracellular tags to visualize surface staining.
Hi Margit!
Thanks for your answer! I have to say, it also seemed to me that there is indeed some spillover but I'm totally new, I tried around but really don't know anything about that; I have additional APC staining (actually Alexa Fluor 594) and FITC.. attached two histograms for you to see..
By the way, put the wrong histograms; here the correct ones.. it is a negative control so I don't have any staining..
Hi Fatam, I gather that you are trying to differentiate dead cells from living once using PI. The correct graphs are the b/w once. You should see PI incorporation once the cells die. However, you can also try Trypan blue, run them using FL 3. the nice part about Trypan blue is that it does not cause any build-up in the SIP or the flow cell. For more information you might want to resort to "Transfusion. 2005 Jul;45(7):1208-13.
Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+ cells after cryopreservation and comparison with trypan blue exclusion staining. Authors: Humpe A, Beck C, Schoch R, Kneba M, Horst HA.
Maybe it could be helpful to "kill" the cells (e.g. heat them up) and make sure, that your PI staining and cytometer settings is able to distinguish living and death cells.
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