TH1/TH2 cytokines post MLR

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Hi:

I would like to look at intracellular Th1/Th2 cytokines by flow in cells
after mixed lymphocyte reactions in addition to measuring cell
proliferation by thymidine incorporation. The cells are from chimeric
mice after mixed bone marrow transplantation. I was thinking of
labelling the irradiated stimulator cells with PKH-26 before running the
one way MLRs for 5 days, so that I could gate out the stimulator cell
populations. I am not too sure how to set up the activation assay for
FACS analysis. Should I restimulate the post MLR responder cells again
with the same stumulators for 4 hrs with golgi stop/golgi plug before
4-color staining for Kb/Kd/CD4/cytokines and analysing on our FACS
Calibur?
 I am hoping the PKH-26 will not interfere in this setup as I am gating
out the positive cells to start with.
Any and all suggestions will be greatly appreciated.

Thankyou

Yasmin

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