TH1/TH2 Intracellular Cytokine Ratio

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.

• Cold specimens (refrigerated or shipped on ice)
• Extensive clotting or hemolysis
•  Specimens older than 48 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

• TNF-α: IL10 (CD3+CD4+) – 13.2 – 30.6
• IFN-γ: IL10 (CD3+CD4+) – 5.8 – 20.5

• Ng SC, Thaker P, Gilman-Sachs A, Beaman KD, Beer AE, Kwak-Kim JYH. Expression of Intracellular Th1 and Th2 cytokines in women with recurrent spontaneous abortion, implantation failures after IVF/ET or normal pregnancy. Am J Reprod Immunol 48:77-86; 2002
• Sacks GP, Clover LM, Bainbridge DR, Redman CW, Sargent IL, Flow cytometric measurement of intracellular Th1 and Th2 cytokine production by human villous and extravillous cytotrophoblast. Placenta 22:550-9; 2001.
• Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Method. 188:117-28; 1995.
• Schuerweigh,A.J., Stevens, W.J.Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta., interleukin-6, and tumor necrosis factor-alpha in monocytes. Cytometry 46:172-176-2001. 

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