Intracellular Cytokine Analysis of Splenic and Ocular Cells
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Corresponding Organization : GlaxoSmithKline (United States)
Variable analysis
- Mouse spleen and eye infiltrating cell cultures
- Intracellular expression of IL-17
- Intracellular expression of IFN-γ
- Intracellular expression of T-bet
- Intracellular expression of RORγt
- Incubation with ionomycin/PMA and Brefeldin A (GolgiPlug, BD Biosciences)
- Staining with conjugated anti-CD3 monoclonal antibody (145–2C11, BD Biosciences)
- Staining with anti-CD4 monoclonal antibody (RM4–5, Biolegend)
- Staining with anti-CD45 monoclonal antibody (30-F11, Biolegend)
- 4% paraformaldehyde fixation and permeabilization (BD bioscience)
- Intracellular staining for T-bet (4B10, Biolegend), RORγt (AFKJS-9, BD Bioscience), IFN-γ (XMG1.2, Biolegend) and IL-17A (TC11–18H10.1, ebioscience)
- Analysis by a MACSQuant Analyzer 10 (Miltenyi Bioitec. Cambridge, MA) and processed by FlowJo (Tree Star, Ashland, OR)
- Exclusion of dead cells using ghost red 780 viability dye (Tonbo biosciences, San Diego, CA)
- Gating of single cells using FSC-A and FSC-H
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