For intracellular cytokine staining, cells were treated with ionomycin (750 ng/ml; Sigma), phorbol myristate acetate (50 ng/ml; Sigma), and BD GolgiStop (1 μl/ml; containing monensin) for 3.5 hours before harvesting. For IL-22 analysis and for cytokine analysis of cultured human CD4+ T cells, BD GolgiPlug (1 μl/ml; containing Brefeldin A) was used instead of BD GolgiStop. Cells were harvested by centrifugation at 400g for 5 min at 4°C, followed by staining with fixable viability stain (BD Horizon FVS700) for 10 min on ice in PBS. Subsequently, surface antigens were stained in FACS buffer for 30 min on ice. Mouse cells were fixed and permeabilized by incubation in BD fixation/permeabilization solution for 15 min at room temperature, followed by washing once in BD Perm/Wash solution. Intracellular cytokines were stained in BD Perm/Wash for 15 min at room temperature. For human cells and for TF staining in mouse cells, the cells were fixed using the fixation/permeabilization buffer in an eBioscience Foxp3 staining kit for 1 hour at 4°C. Cytokines were stained in permeabilization buffer from the same kit for 1 hour at 4°C. Antibodies used for flow cytometry were used at 1:200 dilution, unless otherwise specified. The following antibodies and fluorochromes were used herein: anti–IL-17A (TC11-18H10; BV786 and PE), anti–IFN-γ (XMG1.2; PE-Cy7 and APC), anti-TNF [MP6-XT22; BV650 and fluorescein isothiocyanate (FITC)], anti–IL-22 (1H8PWSR; PE), anti–IL-9 (D9302C12; PE), anti–IL-13 (Ebio13A; PE-Cy7), anti-CD69 (H1.2F3; V450 and Pe-CF594), anti-Foxp3 (FJK-16S; APC), anti-cMAF (symOF1; PE; 5 μl per test), anti-CD103 (M290; BV786), anti-CD4 (GK1.5; BUV395), anti-TCRγδ (GL3; BV421), anti-CD27 (LG.3A10; PE-Cy7), anti-CCR6 (140706; Alexa Fluor 647), anti-CD44 (1 M7; V500), anti-CD19 (6D5; FITC), anti-TCRβ (H57-597; APC-eflour780), anti-Ki67 (B56; BV786; 1:100), anti-IRF4 (3E4; PE), anti-hCD4 (RPA-T4; BB515; 1:25), anti-hCD45RA (HI100; BV605; 1:100), anti-hCD45RO (UCHL1; APC-H7; 1:50), anti-hIL17a (N49-653; BV650; 1:100), anti-hRORγt (AFKJS-9; PE), and anti–hIFN-γ (B27; BV480).

For cell cycle analysis, cells were fixed, after live dead exclusion and surface antigen staining, using the fixation/permeabilization buffer in the eBioscience Foxp3 staining kit for 1 hour at 4°C. Ki67 was stained in permeabilization buffer from the same kit for 1 hour at 4°C. Cells were washed once with permeabilization buffer, and the DNA content was stained using 5 μl of 7-aminoactinomycin D (7-AAD) per test for 10 min on ice in the same permeabilization buffer. Subsequently, FACS buffer was added to the samples and acquired immediately. Apoptosis was measured by staining cells with the Apoptosis Detection Kit (BD) using 5 μl of annexin V–PE and 5 μl of 7-AAD per test and following the manufacturer’s protocol.

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