Do you have any experience with checking expression Granzyme B levels in total human PBMCs using flow cytometry?

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Lora Benoit

What are you using to solubilize the membranes to show intracellular staining? triton x is the best. What fluorochrome are you using to label the GZB? I would use PE as it gives great S/N ratio.

Deepjyoti Kumar Das

Thanks Lora Benoit for your suggesion. I have used FITC labelled GZB. I use BD perm/fix reagent to solubilize membrane (https://www.bdbiosciences.com/en-in/products/reagents/flow-cytometry-reagents/research-reagents/buffers-and-supporting-reagents-ruo/bd-cytofix-cytoperm-fixation-permeablization-kit.554714).

There is also a matter of gating: you will need to gate on the lymphocyte subpopulation on FSC/SCC plot and then do your analysis of granzyme B expression on cd3+ cells ONLY. It is best to use a PE label for intracellular staining.

I have done lymphocyte on SSC/FSC, then live/dead, then CD4 or CD8, and then GZB. Not tried CD3 yet. Need to try this. Thank you for your suggestion.

Thank you @sudeep for your reply. I do use stimulation of antiCD3 + antiCD28 in the PBMCs. Definitely going to try a bright fluorochrome. Right now I was using FITC, which may be one of the reasons I am not getting a window. Regarding 4-1BBL, I am new to this marker. What I found is that, 4-1BBL is more related to NK cells and also it's signalling limits T cell activation (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272921/) . Here, I am trying for T cell activation. If you have any experience/ literature with T cells activation using 4-1BBL please do share.