I a 1 e clone m5 114.15.2

Mice were sacrificed on day 6 or 9 after DSS administration. The colon was cut longitudinally, washed in ice-cold PBS, and cut into pieces that were incubated in pre-digestion solution composed of Hank’s Balanced Salt Solution containing 5% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 5 mM EDTA, and 1 mM dithiothreitol for 30 min at 37°C. Enzymatic digestion was performed in Iscove’s Modified Dulbecco’s Medium (Hyclone, Logan, UT, USA) supplemented with 5% FBS, 1 mg/ml collagenase D (Roche Diagnostics, Indianapolis, IN, USA), and 0.5 mg/ml DNase I (Roche Diagnostics) for 1 h at 37°C with gentle shaking. The cell suspension was passed through a 100-µm nylon mesh and centrifuged. cLP cells were enriched on a 40/80 Percoll gradient (Sigma-Aldrich) (21 (link)). To analyze the components and phenotype of inflammatory cells in the colon, cLP cells were incubated with anti-CD16/-CD32 blocking antibody (clone 93) and then labeled with anti-CD11b (clone M1/70), anti-F4/80 (clone BM8), anti-CD206 (clone C068C2), anti-Dectin-1 (clone RH1), anti-CD11c (clone N418), anti-major histocompatibility complex (MHC) II (I-A/I-E, clone M5/114.15.2), and anti-Gr-1 (clone RB6-8C5) antibodies (all from Biolegend). Antibodies were used at 1:100 dilution. The cells were sorted on a Canto II cytometer, and data were analyzed with FlowJo-X software.

Blood samples from Foxp3^YFP/Cre+/−tdTomato^+/− and IL17a^Cre+/−tdTomato^+/− reporter mice were prepared for flow cytometry as previously described (38 (link)). Zombie Aqua stained, FC receptor blocked blood cells were stained with anti-mouse CD11b (clone M1/70), F4/80 (clone BM8), Ly-6G (clone 1A8), B220 (clone RA3-6B2), and I-A/I-E (clone M5/114.15.2) fluorochrome conjugated mAbs from BioLegend. Cells were acquired on an LSR II flow cytometer (BD Biosciences, San Jose, CA) and fluorescence emissions analyzed with FlowJo software (Tree Star, Ashland, OR).