Measurement of CD4+CD25+Foxp3+ Treg cells by flow cytometry

Cells from intestinal PPs were resuspended in 3% FCS/PBS solution at a final cell concentration of 1 × 10⁶-10⁷/mL. The cell suspension (n = 8) was incubated for 30 min with APC conjugated anti-rat CD4 antibody (RM4-5) and PE conjugated anti-mouse CD25 antibody (PC61.5)at 37 °C in dark. Cells were centrifuged at 5000 rpm at 4 °C for 2 min, fixed in Fix/Perm Buffer (eBioscience, San Diego, CA, United States) for at least 1 h at 37 °C, and then incubated with FITC conjugated anti-mouse Foxp3⁺ antibody (FJK-16s) for 30 min at 37 °C in the dark. Cells labeled with PE conjugated rat IgG2a were used as the isotype negative control. Rate of CD4⁺CD25⁺Foxp3⁺Treg cells was analyzed using FACS Calibur (BD Biosciences).