Fixation permeabilization buffer kit

PBMCs were stimulated with anti-CD3 antibody (100 ng/ml) for 6 h. Then, after 1 h of incubation, brefeldin A (GolgiPlug, BD Biosciences) and monensin (GolgiStop, BD Biosciences) were added to the culture to cause intracellular cytokine accumulation. Cells were surface-stained with anti-CD3-Horizon V500, anti-CD4-PE-Cy7, anti-CD8-APC-H7, anti-CD28-APC, and anti-CD57-Pacific blue. Then, cells were fixed and permeabilized with the Fixation/Permeabilization Buffer Kit (BD Biosciences). Cells were then stained to detect intracellular cytokines with anti-TNF-PE-Cy7 and anti-IFN-γ-FITC (all from BD Biosciences). FACS analysis was performed with an LSR II Flow Cytometer, and data were analysed with FlowJo software.

The phenotype of colon-infiltrating Tregs was determined by flow cytometry [17 (link)]. Briefly, about 1 × 10⁶ cells per sample were incubated with antihuman CD4 and IL-10 antibody conjugated with fluorescein isothiocyanate (FITC; BD Biosciences, Franklin Lakes, NJ, USA), phycoerythrin (PE; BD Biosciences), peridinin chlorophyll A protein (PerCP; BD Biosciences) or allophycocyanin (APC; BD Biosciences). For the intracellular staining, cells were previously stimulated with phorbol myristate acetate (PMA) and ionomycin for 4 h at 37 °C with the addition of 1 μg/mL Golgi plug. Intracellular staining for forkhead box P3 (Foxp3) was performed using the BD Bioscience fixation/permeabilization buffer kit. Flow cytometric analysis was conducted on a BD Biosciences FACSCalibur and analyzed by using the flowing software analysis program.