Hej,
we are struggling with T-Bet flow cytometry staining for lymphocytes from human colorectal cancer tissue. We are getting a good staining from PBMCs but no positive cells from the tissue. We have tried different kits for transcription factors (ebioscience, BD) as well as different antibody clones. Furthermore, we have exposed the PBMCs to the same digestion enzymes like the tissue cells and T-Bet is still detectable. Any suggestion about a different protocol or Fixing/ staining times/stimulation? Thanks!
From what you describe, seems like the problem is not with your digestion/staining protocol. Could it be that you don't have measurable levels of T-bet expressing cells in your cancer tissue? Tumor microenvironments are supposed to be immunosuppressive and it could be that you don't have many Th1 cells in there. If you are just looking at functional Th1 responses, an alternate approach would be to do ELISPOT, which is much more sensitive. Usually, most of the spots you see are from Th1 cells secreting IFN-g. But you won't be able to correlate the signal specifically to T-bet though.
Thanks for your answer. I guess we will need to go with another method too.
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