I want to analyze the following T regulatory cell phenotypes: CD4+CD25+IL-10+ T regs, CD4+CD25+Foxp3+ T regs, and CD4+LAP+ (TGF-beta1-secreting) T regs.
The IL-10+ and LAP+ T regs seem to have to be stimulated under different conditions and with different reagents. Also Foxp3+ and IL-10+ T regs both have to be intracellularly stained, and require different commercially available permeabilization/fixation buffer sets, as per several published protocols in literature.
I will be isolating these cells from spleen and lymph nodes.
I am leaning towards making 3 separate aliquots of 1 x 10^6 cells per organ, and staining each one million cells to identify separate population of T regs. For example, the first million cells, I will only stimulate and stain with CD4, CD25 and IL-10. The second million cells I will only stain with CD4, CD25 and Foxp3, and so on...
Would you recommend this kind of approach, or have any other suggestions/advice?
Also, should I stimulate the cells with antigen I used to immunize mice, or with solutions that will guarantee secretion of cytokines (i.e. PMA/Ionomycin,BFA for IL-10 cells, and CD3/CD28,IL-2 for LAP+ cells)?
Much appreciated.
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