CST, 28572S, Human T Cell Th1 Cytokine Response Flow Cytometry Panel

Assay Kit for studying CD3epsilon/IFN-gamma/CD8A/CD4/TNF-alpha in the research area. Product Usage Information 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 CD3 epsilon Activating (OKT3) Mouse Monoclonal Antibody (Low Endotoxin, Azide-free) # 92511 , 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 Triton TM 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 Fixable Viability Dye # 59863 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. Storage 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 NaH 2 PO 4 , 150 mM NaCl, 0.09% NaN 3 , 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 4 o C. 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. Protocol Available protocols: Flow Cytometry (Fixed/Permeabilized) Specificity / Sensitivity Each antibody in the Human T Cell Th1 Cytokine Response Flow Cytometry Panel detects endogenous levels of its target protein. CD3 (UCHT1) Mouse Monoclonal Antibody (PE Conjugate) recognizes CD3ε. CD3 (UCHT1) Mouse Monoclonal Antibody (PE Conjugate), CD4 (RPA-T4) Mouse Monoclonal Antibody (violetFluor 450 Conjugate), and CD8 alpha (RPA-T8) Mouse Monoclonal Antibody (PE-Cy7 Species Reactivity: Human Source / Purification Monoclonal antibodies were purified from tissue culture supernatant via affinity chromatography. The purified antibodies were conjugated under optimal conditions, with unreacted dye removed from the preparation.