The ratio of TH1:TH2 cytokines in the cytoplasm of CD3+CD4+ lymphocytes can be determined by flow cytometry. Lymphocytes (mononuclear cells) are stimulated with phorbol myristate acetate (PMA) and ionomycin in the presence of GolgiPlug (brefeldin A), a Golgi transport inhibitor, for 18 hours. The cells are then harvested, permeabilized, and stained with phycoerythrin–conjugated anti-cytokine antibodies specific for TNF-α, IFN-γ, or IL-10. Four-color flow cytometry is used to determine the percentages of intracellular cytokines in CD3+CD4+ lymphocytes.
Stimulation down-regulates CD4 molecules on the cell surface and prevents their reliable detection. Therefore, CD3+CD4+ cells are identified by negative gating using ECD-anti-CD3 and FITC-anti-CD8. Lymphocytes that are CD3+CD8− represent the CD3+CD4+ population. The percentages of cytokine-positive CD3+CD8− cells are measured for each of the three cytokines, and ratios are calculated for TNF-αCD3 and FITC-anti-CD:IL-10 and IFN-γ:IL-10.
Women with recurrent spontaneous abortion may have higher TH1:TH2 ratios than women with a normal pregnancy history.
Send at room temperature. Do not refrigerate. Deliver to the laboratory within 24 hours.
If a specimen exceeds 48 hours, lymphocytes will be isolated and viability assessed. If viability is >80%, the assay will be performed; if viability is <80%, the specimen will be rejected.
Flow Cytometry