Cd4 pe cy7

Isolation of lamina propria (LP) lymphocytes was performed according to adapted protocols of Weigmann et al. 13 (link) and Geem et al. 14 Briefly, the ileum and colon were harvested and opened longitudinally, washed to remove fecal content, and shaken in HBSS containing 5 mM EDTA and 5% FBS for 20 min at 37 °C twice. The epithelial cell suspension was discarded and the remaining intestinal sections were cut into small pieces and incubated in HBSS containing 5% FBS, 0.5 mg mL -1 collagenase VIII (Sigma-Aldrich, St Louis, MO), and 40 μg mL -1 DNase I (Roche Diagnostics, Basel, Switzerland) for 20 min at 37 °C followed by vortexing. The digested tissues were washed twice with HBSS. The collected cells were then resuspended in staining buffer containing PBS, 2% FBS and 0.1% NaN 3 and stained for surface CD4-PE-Cy7 (clone: RM4-5, Tonbo Bioscience, San Diego, CA) and CD25-FITC (7D4, BD Bioscience, San Jose, CA). Intracellular staining of Foxp3-PE (FJK-16s, eBioscience) was performed using a Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer's protocol. Separately, LP lymphocytes were stained for surface CD11c-PE (N418, Tonbo Bioscience) and CD103-PE-Cy7 (2E7, Biolegend) for CD103 + DCs analysis. Samples were analyzed by flow cytometry using BD FACSVerse and BD FACSSuite software (BD Bioscience).