Apc conjugated anti cd4 antibodies

Mice were sacrificed, and intestines were removed, opened longitudinally, and cut into 1cm pieces. The pieces were then incubated twice in 5 mM EDTA in PBS for 15 min at 37°C, and then the epithelial cell layer was removed by vortexing and passing through a 100-µm cell strainer. After incubation with EDTA solution, tissues were washed, minced into small pieces, and digested for 1 h at 37°C in digestion solution containing 4% FBS, 0.5 mg/ml collagenase D (Roche), 0.5 mg/ml DNase I (Sigma-Aldrich), and 3 mg/ml dispase II (Sigma-Aldrich). The resulting solution was strongly vortexed and passed through a 40-µm cell strainer. Mononuclear cells were isolated by gradient centrifugation on a 30/70% Percoll gradient (GE Healthcare) at 670 g for 30 min. Isolated cells were stained for ILC3 isolation or stimulated with PMA (50 ng/ml), ionomycin (750 ng/ml), and Brefeldin A (BioLegend) for 4 h and then stained with PerCP/Cy5.5-conjugated IL-17A, and APC-conjugated anti-CD4 antibodies. For ILC3 isolation, cells were stained with FITC-conjugated anti-Lineage cocktail, APC-conjugated anti-CD117, and PerCP/Cy5.5-conjugated anti-NKp46 antibodies, and then Lineage⁻ NKp46⁻ CD117⁺ cells were sorted. All fluorochrome-conjugated antibodies were purchased from BioLegend.