Intracellular staining for transcription factors in human dendritic cells

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happyapple82
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Intracellular staining for transcription factors in human dendritic cells

Hi,
I am new to here, and I am now trying to stain for T-bet or Foxp3 in human dendritic cells after transduction.
I used the eBioscience Foxp3 intracellular staining kit (the buffer set); and I tried different perm/fix and staining time.
I still cannot get the stainng work, there is only once that I see a 50% postitive events, there dendritic cells are transduced with Ad.T-bet, or Ad.Foxp3, normally there should be around 50% T-bet positive cells after transduction, however, I can only get around 3%-10% positive cells, which is not reflecting true efficiency of transduction.
Anyone has experience on Intracellular staining for nucleus protein in human DC? Any suggestion?

Thank you very much!

samm
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Use a nuclear staining

Use a nuclear staining control - such as H2A or HMGB1 as an intrac staining positive control. I've used the eBio system, and it works pretty well, though we now use our own 'kit'. (protocol on this site). Are you performing an o/n staining for Foxp3?
Does your Ad-Tbet/Foxp3 have a reporter built in - you need to ensure that transduction followed by transcription is occuring. (i.e. how are you gauging efficiency?)

happyapple82
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To Samm:

To Samm:

Thank you for your suggstion. The transduction efficiency was also tested by PCR, although it cannot give the exact number of positive population.  We also tested the efficiency by tranduce tumor cell lines.  Those adenovirus seems to work well.
ebioscience kit sometimes work for me, but most times not. So I am eager to find another stable method that I can get most staining working.
I am not sure wether it is some thing related with the human dendritic cells? I know the kit work well on T cells and mouse cells.

Still cannot figure out which step went wrong, any way, I will try to set  a control in my exp to make sure.

Thanks!

samm
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Hi! If you want to try your

Hi! If you want to try your own, initially surface stain as usual for DCs (CD11c, MHCII, CD86 etc) if you want. Wash well, resuspend in minimal volume, fix with 4% neutralized PFA or 10% neutral buffered formalin at RT for ~30 mins. Permeabilize with excess volume of 0.25% saponin+0.05%TX-100 in PS/HBSS containing 0.5% low Ig serum for 15 min. (You can pre-prepare everything, but add the saponin immediately prior to staining). Wash 2x to ensure no trace of fixative remains.
Add your anti-Foxp3 Ab in the perm buffer - you can incubate this o/n in the refrigerator.
Wash well, resuspend and acquire.
(Your cells alone ctrl with all steps but Ab addition are critical since autofl increases)

happyapple82
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Thank you very much! Samm,

Thank you very much! Samm, just want let you know your method worked for my experiments.
Maybe you can post it as a specific protocol for human DC transcription factor in the forum.
I really appreciate your help a lot!

Jessica Xu
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Hi,I am a gradstudent from

Hi,I am a gradstudent from China,and I also met some problem in FOXP3 staining.Firstly,the non-specific background staining is high;secondly,the CD4+FOXP3+ cell is so low.And I happen to see your protocol,I used to use our own Perm buffer kit and it is different from yours. It is constitute of  0.15%saponin+1%BSA in 1xPBS.And I usually let cell permealbilize in 100 ul in 10mins,do you think the perm buffer and the time is OK?
thank you for your any reply.