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.