Usually the pattern of Th response are evaluated by flow cytometry determining the expression of surface markers. This method quantifies the number of cell with certain phenotype. But also the expression of master regulators is lineage characteristic. So I ask if it´s possible to determine the Th pattern by expression and its degree of master regulators by Western blot instead the classical surface markers by flow cytometry.
I don't do many westerns but I suppose you could potentially look for GATA 3, RORyT, T-bet and FoxP3 activation, although looking at what the cell is actually making might be better! Try culturing them with anti CD3 and 28 - then doing ELISAs for IL-10, IFNy, IL4, IL-17 - then you can also do westerns so you have two tests on one batch of cells?
Only small part of clones responds to given antigen at the same time. So, your Western blot analysis requires preliminary isolation of these clones to be correct. Otherwise, you will detect thousands of responses, which have no relation to the studied antigen.
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