I try to establishment a Panel TH1 cells by surface antigen CXCR3 + CCR6- and transcription Factor T-bet.
But the problem is always the percentage of T-bet low(3-4%)
I hope someone can help me
Hi Michael,
We stain for T-bet and other transcription factors quite often. What protocol are you using for your intracellular staining? We found staining much improved by using a FoxP3 staining kit. They're available from BD and eBiosciences
Hi, I totally agree with Michael! It makes such a big difference using the different specialized buffers: personally I prefer the eBiosciences "FoxP3" buffers...
I agree with the previous responses: using commercial kits can sign. improve the quality of transcritpion factor staining for FACS, e.g. the BD Fix/Perm buffer kits.
However, 3-4% T-bet+ cells might be not sooo low in first place ... depending on the condition were you get your cells from (organ, stimulated/primed in vivo?) this might actually be quite realistic.
Depending on the source of the cells, you could gate on a population which is known to express Tbet at higher level (NK cells) to check the quality of your staining. Running a PBMC sample may be helpful.
Thank you for your reply
I used BD Fix / Perm buffer kits.
I isolated cells from whole blood,and I used CXCR3+CCR6- INFG+ TBET to detect TH1 but I found the percentage of TH1 with These Markers sooo low
I'm afraid that BD Fix/Perm buffer is not enough strong for Tbet staining. We use the BD buffer for transcritpion factors and works quite well.
Thank you for your reply,
Detecting the transcription factors, for example, T-bet, gata 3, RORG, needs stimulation or without Stimulation
you can detect them even without stimulation. But the right buffer is needed. I also tried several time with normal cytofix/cytoper and had no results
I did the same thing, i stimulated T cells for 48 hours and then detected the transcription factors. I had also about 5 % of T cells positive for T bet in controls unstimulated T cells, but with stimulation we found an increase (15 % of positive cells). The percentage of t cells positive for T bet in controls cells depend on the state of the patient (if he is a patient with chronic inflammation, then you can see an increase in T bet). To improve your staining, you can increase the incubation time (usually it is 30 min but you can try 45 min or 1 hour).
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