Characterizing Colon-Infiltrating Regulatory T Cells

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.