The TH1:TH2 cytokine ratio in the cytoplasm of CD3âșCD4âș 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 CD3âșCD4âș lymphocytes.

Because stimulation causes down-regulation of CD4 surface expression, CD3âșCD4âș cells are identified using negative gating with ECD-anti-CD3 and FITC-anti-CD8. Lymphocytes that do not bind CD8 (CD3âșCD8⁻) represent the CD3âșCD4âș subset. The percentage of cytokine-positive CD3âșCD8⁻ 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 CD3âșCD4âș lymphocyte activation.

Send at room temperature. Do not refrigerate.

• Cold samples (due to refrigeration or shipment on ice)
• Extensive clotting
• Hemolyzed samples
• Specimens >48 hours old

If a specimen is >48 hours old, lymphocytes will be isolated and viability assessed.

• If viability ≥80%, the assay will be performed.
• If viability <80%, the specimen will be rejected.

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.

• 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.
• determination of interleukin-1 beta., interleukin-6, and tumor necrosis factor-alpha in monocytes. Cytometry 46:172-176-2001.

Back to Reproductive Immunology