I isolate PBMCs from human blood and try to perform intracellular staining for T-bet (i'm interested in CD19+ cells, but for now i use CD3+ as a control). I do know there are kits that are specific for transcription factors, but i own the cytofix/cytoperm kit and i gave it a try (besides, Y Liu et al. 2017 - and others maybe - performed the staining using the very same kit). Once gating the CD3 cells, there is a very low T-bet+ cells percentage. As the majority of T cells belongs to the CD4+ compartment, i expected a high percentage - more than 20% actually... Any ideas/suggestions??
Dear Athanasie, I had tried to combine intracellular and intranuclear staining together in the past using either the BD Cytofix kit or the eBioscience FoxP3 kit, but it never worked for both targets (transcription factor and cytokine) at the same time... In my case it was not T-bet but Eomes that I wanted to stain for, but I assume it is similar with other transcription factors like T-bet, RORgt, GATA-3 etc. So, the obvious solution is to go with the eBioscience FoxP3 kit that is optimized for intranuclear staining, however, something that you could also try is using "homemade" buffers instead. You could try for instance Triton X-100 or Tween 20 diluted in PBS 1X as permeabilization buffers. Personally, I used to do this to stain fixed tissue sections, but I have found people in ResearchGate that have used these buffers to successfully stain for intranuclear targets in flow cytometry too, so you could also try, eg 0.2% Tween 20 in 1X PBS as permeabilization buffer.
As fixative you could also try formaldehyde vs paraformaldehyde, since there are some differences there too.
I hope it helps a bit!
Good luck,
Michalis
Dear Athanasios Sachinidis
For transcription factor stains I had good results (despite not for T-BET), but for FOXP3 using BD Bioscience's FOXP3 staining reagents.
They also have a little tutorial presentation that links to more resources like the BDFACSselect database that outlines buffer compatibility. Maybe that helps. Please see below.
https://www.bdbiosciences.com/documents/webinar_022713_intracellular_flow.pdf
All the best,
Michael
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