Live dead fixable blue dead stain kit

Before staining, all cells were treated with Fc-block (anti-CD16/CD32, clone 93, 1μg/ml, Biolegend, San Diego, USA) in PBS, for 10min, on ice. Cells were washed and stained in HBSS + 2% HS on ice for 30min. After staining the cells were washed and fixed using the BD Cell Fix kit (BD Biosciences, USA). For all analyses, dead cells were excluded using viability dyes (LIVE/DEAD fixable blue dead stain kit, Thermo Fisher Scientific, Carlsbad, USA, Zombie Aqua fixable viability kit, Biolegend, San Diego, USA). Fluorescence-activated cell sorting (FACS sorting) was performed on a BD ARIA3 (BD Biosciences, USA) or a Moflo Astrios (BD Biosciences, USA) instrument. For flow cytometry analysis, the samples were acquired on a BD Sorp LSR2 (BD Biosciences, USA).

Excised colons were cleared of fecal matter, cut longitudinally and rinsed in ice cold PBS before transfer to PBS containing 2% FBS on ice. Tissue was subsequently transferred into PBS containing 2 mM EDTA and 1 mM DTT then incubated for 15 min at 37°C, shaking at 180 rpm, then filtered (70 μm). The tissue was incubated in a digestion enzyme cocktail (Collagenase V (0.85 mg/ml); Collagenase D (1.25 mg/ml); Dispase II (1 mg/ml) and DNase (30 μg/ml)) for 25 min, before vortexing, filtering (40 μm) and re‐suspending cells. Cell staining was carried out in a 96‐well V‐bottomed plate. After live/dead staining (LIVE/DEAD fixable blue dead stain kit, ThermoFisher Scientific) and blocking (1:100 anti mouse CD16/CD32, Fisher Scientific, UK) cells were stained with a panel of antibodies (Table S2). All extracellular antibodies were utilized at 1:200 and intracellular at 1:100. Amine Reactive Compensation beads (Thermo Fisher Scientific) were utilized with the live/dead stain for compensation controls. One Comp eBeads (ThermoFisher Scientific) were utilized to produce single stained compensation controls. Samples were analyzed on a BD LSR Fortessa Flow cytometer and data collected using FACSDiva software. Data analysis was performed using Flow Jo Version 10.7.1.