Collagenase type 4 and dnase 1

To obtain LP cells, murine colons were cut into small segments (1-cm long), washed with Dulbecco's Phosphate-Buffered Saline (DPBS) 1× (Gibco, Waltham, MA) + 2.5 mmol/L EDTA (Ambion, Thermo Fisher Scientific) to separate from epithelial cells, and digested with collagenase type IV and DNase I (Sigma Aldrich) for 30 minutes at 37°C after GentleMACS processing. Resulting cell suspensions were pelleted by centrifugation, washed with DPBS 1× + 0.5 mmol/L EDTA, and passed through 100-μm and 30-μm cell strainers (Miltenyi Biotec, Bergisch Gladbach, Germany) and washed with DPBS (Gibco) + 0.5% bovine serum albumin (BSA; Sigma-Aldrich).
MLNs were isolated from mice treated with anakinra or vehicle. MLNs were passed through a 30-μm cell strainer (Miltenyi Biotec) to obtain a single-cell suspension and washed with DPBS (Gibco) + 0.5% BSA (Sigma-Aldrich).

The intestines of 16-week-old Winnie and WT mice were removed and tissue digested to obtain a single-cell preparation. Colons were cut into small segments (1 cm long), washed with DPBS 1X (Gibco, Waltham, MA, USA) + 2.5 mM EDTA (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) to remove epithelial cells, and digested with collagenase type IV and DNase I (Sigma Aldrich, St. Louis, MO, USA) using the GentleMacs suggested protocol for 30 minutes at 37°C. Resulting single-cell suspensions from colonic lamina propria (LP) were pelleted by centrifugation, washed with DPBS 1X + 0.5 mM EDTA, and passed through 100-μm and 30-μm cell strainers (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were then washed with DPBS 1X + 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and labeled with CD45.2-FITC and MHC II-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Finally, cell suspensions were labeled with 7-AAD Staining Solution (Miltenyi Biotec, Bergisch Gladbach, Germany) to distinguish between viable and dead cells, also according to the manufacturer’s instructions. Flow cytometer data analysis was performed using NAVIOS software (Beckman Coulter, Brea, CA, USA), with at least 3 experiments performed.