I have used PE and PE-Dazzle 594 fluorochromes and have managed to get NK cells to properly show GranzymeB expression after 4 hr PMA/ionomycin stimulaton, but for some reason my CD8 cells in the same tube never show expression compared to appropriate isotopes. This is all in the same tube, mind you. I have tried without stimulation, I have tried using splenocytes from both naive and tumor-bearing mice, I have tried 2 different clones of the antibody. Nothing seems to work in my hands. However, in the same tubes, T cells and NK cells show IFNy and TNFa expression, so the problem is not with permeability of the cells after fixation. Please let me know any suggestions you have!
Dear Austin McMasters
I briefly wanted to reach out concerning your inquiry. I would presume that the kinetics of granzyme expression differs between NK and CD8 positive T cells, especially as granzyme b is an early activation marker for NK cells
I have done similar experiments in the attached paper and usually restimulated CD8 T cells for 24 hours before FACS analysis. Please check experiments and reagents specifically in Figure 4.
Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...
For a more detailed analysis of the kinetics of granzyme expression in mixed 129/SvJ mice, please consider
Article Differential Expression of Granzyme B and C in Murine Cytoto...
And for differences in the regulation of protein expression between NK cells and CD8 T cells, please see
Article Differential Regulation of Granzyme B and C Expression in Mu...
I hope that helps.
All the best & good luck with your experiments,
Michael
As an alternative to Granzyme B, you could try staining CD107a (Lamp1). CD107a is a marker for degranulation and is often detected at the surface of the CTL upon activation confirming degranulation of GranzymeB and perforine. So, It is good indicator of functional cytotoxicity.
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