The TH1:TH2 cytokine ratio in the cytoplasm of CD3⁺CD4⁺ lymphocytes is determined by flow cytometry. Mononuclear cells are stimulated with phorbol myristate acetate (PMA) and ionomycin in the presence of GolgiPlug (brefeldin A), a Golgi transport inhibitor, and prednisolone, a corticosteroid that inhibits inflammatory cell activation, for 18 hours.
Following stimulation, cells are harvested, permeabilized, and stained with phycoerythrin-conjugated antibodies to TNF-α, IFN-γ, or IL-10. Four-color flow cytometry is used to identify intracellular cytokine expression within CD3⁺CD4⁺ lymphocytes.
Because stimulation causes down-regulation of CD4 surface expression, CD3⁺CD4⁺ cells are identified using negative gating with ECD-anti-CD3 and FITC-anti-CD8. Lymphocytes that do not bind CD8 (CD3⁺CD8⁻) represent the CD3⁺CD4⁺ subset. The percentage of cytokine-positive CD3⁺CD8⁻ cells is determined for each cytokine, and ratios are calculated for TNF-α:IL-10 and IFN-γ:IL-10.
Results from this assay are compared to those from the TH1:TH2 Intracellular Cytokine Ratio test performed without prednisolone to assess corticosteroid-mediated inhibition of CD3⁺CD4⁺ lymphocyte activation.
Send at room temperature. Do not refrigerate.
If a specimen is >48 hours old, lymphocytes will be isolated and viability assessed.
Deliver to the laboratory within 24 hours.
Flow Cytometry
The TH1:TH2 Intracellular Cytokine Ratio with Prednisolone may only be performed in conjunction with the TH1:TH2 Intracellular Cytokine Ratio test.