Foxp3 intracellular staining kit

Stomach intraepithelial leucocytes were prepared as described above. Cells were incubated with ZombieFITC (1:500, BioLegend) for dead/live discrimination and 10 μg ml⁻¹ anti-CD16/32 antibodies (39, BioLegend) to block Fcγ receptors for 15 min at room temperature. After washing with PBS supplemented with 5% FCS (Sigma-Aldrich), cells were stained with CD45 BV785, CD117 APC and IgE BV421 (R35-72, BD Bioscience) for 20 min on ice and protected from light. After washing and centrifugation, cells were fixed and permeabilized using a FoxP3-intracellular staining kit (BioLegend) according to the manufacturer’s instructions. Cells were stained with 0.11 μg ml⁻¹ anti-5-HT (5HT-H209, Dako) or isotype control antibodies for 30 min, washed in PBS and stained with anti-mouse-IgG1 (RMG1-1, BioLegend) antibodies for 30 min before analysis with a BD LSRFortessa (Becton Dickinson).

For intracellular cytokine staining, cells were re-stimulated for 4 h before harvest with phorbol myristate acetate (PMA) (50 ng/ml), ionomycin (1 μM) (both from Sigma-Aldrich), and Brefeldin A (BioLegend). Then, the cells were harvested, fixed, and permeabilized using an intracellular staining kit (eBioscience) prior to staining with PerCP/Cy5.5-conjugated anti-IL17A (506,919, BioLegend) and PerCP/Cy5.5-conjugated anti-Ki-67 (652,423, BioLegend) antibodies.
For transcription factor staining, cells were harvested directly, fixed, and permeabilized using a FOXP3 intracellular staining kit (BioLegend). Cells were then stained with a FITC-conjugated anti-Foxp3 antibody (11–5773-80, eBioscience) or an APC-conjugated anti-Foxp3 antibody (17–5773-82, eBioscience). Cells were also stained with APC-conjugated anti-CD152 (106,309, BioLegend), FITC-conjugated anti-GITR (120,205, BioLegend), PE-conjugated anti-ICOS (313,507, BioLegend), PE-conjugated anti-CD25 (102,008, BioLegend), and PerCP/Cy5.5-conjugated anti- programmed death-1 (PD-1; 135,207, BioLegend) antibodies. Stained cells were analyzed using an Accuri C6 Plus flow cytometer (BD Biosciences) or a FACSCalibur flow cytometer (BD Biosciences).

For intracellular staining, indicated populations of T cells were fixed and permeabilizied with Fixation/Permeabilization buffer set (00–5523; eBioscience), washed, and stained (30 min, 4 °C) with primary antibodies (PKC-θ or Dlgh1). Then, the cells were incubated (30 min, 4 °C) with a FITC-conjugated secondary antibodies (Jackson ImmunoResearch Lab. Inc., West Grove, PA). Human-specific antibodies used for flow cytometry CD4 (clone RPA-T4) and CD25 (M-A251), were purchased from eBioscience, while FOXP3 (clone 249D) is from BioLegend. Cells were stained for CD4, CD25, FOXP3 using the BioLegend FOXP3 intracellular staining kit. We analyzed samples in a FACSCalibur machine (BD, Franklin Lakes, NJ).