we are doing flow cytometric analysis of mouse T lymphocytes. We want to check the nuclear transcription factors. Currently we are running low on PMA and ionomycin. Is it possible to do the analysis without them?
Hello John!
Thanks for your generous response. Actually we are analyzing the transcription factors for the first time. The protocol we found are not very clear about the usage of PMA/ionomycin. So we thought if we could omit the step.
Hello Sreyasi,
This would depend on your experimental setting. If you are working with naïve T cells, then you would need to stimulate them in order to evaluate the expression of the transcription factors. However, antigen-experienced T cells may constitutively express the transcription factors you are looking for and you would not need to perform the stimulation. For example, th1 cells express t-bet, Tregs express FOXP3, th17 cells express RORg, and so on. Thus, you need to investigate in the literature if the transcription factor(s) you are looking requires T cells to be stimulated.
Best regards,
Heriberto
Hi!
Why not? If CD4 T-cells undergo differentiation (for example, in the intestine) they will express different transcription factors. Tregs express FoxP3 without any stimulation and FoxP3 is readily detectable. The other TFs may be also detectable. Another question is that few cells will be differentiated in normal conditions.
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