I need help to analyze the FACs results, which were performed on B cells to assess the cell survivability. I used PI and 7-ADD dyes and I saw two distinct cell populations in the SSC vs. FS blot. I am expecting to see alive cells in the majority of the cell population. I have problems with gating since I don't know the morphology of dead or alive B cells as well as the debris. Ideally, high side scatter and low forward scatter are the phenomena for the dead cells. When I change the gating based on that the percentage of the alive or dead cells are changing. So, I really need to do ‘gating’ properly. I also wanted to include a positive control in which I tried to kill the cells. I exposed the cells to UV and I did cold-heat shock, I tortured the cells basically. But since then, positive and actual samples showed the same cell population, it seems it didn’t work. This is another question, how can I kill the cells without lysing them? Another thing I couldn’t get is since I used dyes which stain the dead cells (PI and 7 ADD), how is that still possible to see alive cells? In the attachment, you can see results from PI staining and the positive control for PI staining. (The 7-ADD staining gave the same results).
Try some Etoposide or Staurosporine from Sigma to induce Apoptosis. This could take from 12-24hrs and could form the basis of a positive control for dead cells not made by freeze thaw. Alternatively, if these are A20 or something similar maybe FAS-FASL agonism could kill them. // Regarding your FS/SS and PI and 7-AAD stainings. I think it is reasonable that your cells not in Gate1 could stain positive. They could be undergoing death as you process them and stain positive even though the FS/SS characteristics are those of "Living" cells. Be conservative with your gatings.
When we use flow cytometry to study suspension cells, we usually gate the main cell population in FSC-SSC plot. In your data, it seems that you did not gate the main cell population but gate the minor one. The black dense cell population in FSC-SSC should be the main cells you may gate, in which most of your normal cells are located.
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