Human T Cell Th1 Cytokine Response Flow Cytometry Panel

Cytokine production by T cells can be induced by activating cells through a variety of methods. These include stimulation through the T cell receptor by incubation with an agonist CD3 antibody such as Human CD3ε Activating (OKT3) Mouse mAb (Low Endotoxin, Azide-free), or treatment with a combination of PMA (phorbol-12-myristate-13-acetate) and Ionomycin. The addition of Brefeldin A and Monensin during the cell stimulation will limit cytokine secretion and facilitate detection of intracellular cytokines. All antibodies in this kit are compatible with the Flow Cytometry TritonTM X-100 Permeabilization Protocol for Directly Conjugated Antibodies and can be used in a single staining mix on fixed and permeabilized cells. Prior to fixation and antibody incubation, we recommend adding a fixable viability dye such as the Ghost Dye^™ Violet 510 Viability Dye to enable identification and exclusion of dead cells from analysis.

Gating strategy for observing production of Th1 cytokines by T cells:
If a fixable viability dye was used, first gate on viable cells. Next, gate on lymphocytes based on forward scatter and side scatter. To observe cytokine production by helper T cells, gate on the CD3+CD4+ cells within the lymphocyte gate and then identify cells within this gate that are positive for staining of IFN-γ and/or TNF-α. To observe cytokine production by cytotoxic T cells, gate on the CD3+CD8+ cells within the lymphocyte gate and then identify cells within this gate that are positive for staining of IFN-γ and/or TNF-α.

CD3 (UCHT1) Mouse mAb (PE Conjugate), CD4 (RPA-T4) Mouse mAb (violetFluor^™ 450 Conjugate), and CD8α (RPA-T8) Mouse mAb (PE-Cy7^® Conjugate) are supplied in 10 mM NaH₂PO₄, 150 mM NaCl, 0.09% NaN₃, 0.1% gelatin, pH7.2. TNF-α (D1G2) Rabbit mAb (Alexa Fluor^® 647 Conjugate) and IFN-γ (D3H2) XP^® Rabbit mAb (Alexa Fluor^® 488 Conjugate) are supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4oC. Do not aliquot the antibodies. Protect from light. Do not freeze.

All components in this kit are stable in accordance with the date printed on the outer packaging label when stored at the recommended temperature. Please refer to product labels, datasheets, or web pages for specific "Best By" dates for each individual component.

Each antibody in the Human T Cell Th1 Cytokine Response Flow Cytometry Panel detects endogenous levels of its target protein. CD3 (UCHT1) Mouse mAb (PE Conjugate) recognizes CD3ε. CD3 (UCHT1) Mouse mAb (PE Conjugate), CD4 (RPA-T4) Mouse mAb (violetFluor^™ 450 Conjugate), and CD8α (RPA-T8) Mouse mAb (PE-Cy7^® Conjugate) detect epitopes within the extracellular domains. TNF-α (D1G2) Rabbit mAb (Alexa Fluor^® 647 Conjugate) and IFN-γ (D3H2) XP^® Rabbit mAb (Alexa Fluor^® 488 Conjugate) detect epitopes within the intracellular domains.

The Human T Cell Th1 Cytokine Response Flow Cytometry Panel can be used to observe production of signature Th1 cytokines including IFN-γ and TNF-α.

T cells are identified by expression of CD3. There are two major subsets of conventional T cells: helper T cells which express CD4, and cytotoxic T cells which express CD8. Th1 cells are one of several subsets of helper T cell subsets. Th1 cells promote cell-mediated immunity to combat viral infection, intracellular bacteria, and tumor cells. Both Th1 and cytotoxic T cells can produce IFN-γ and TNF-α.