Isolation and Characterization of Colonic and Splenic Immune Cells

Colons were collected, flushed and enzymatically processed as previously described (2 (link)). Briefly, minced distal colons were washed 3 times for 20 min at 37^oC in 2mM EDTA, 10% FCS, 25mM Hepes, HBSS buffer and subsequently digested using a Liberase/DNAse 1 (Sigma-Aldrich, St Louis, MO). Mononuclear cells were isolated by Percoll gradient separation (GE Healthcare Life Science, Pittsburgh, PA). Splenocytes were isolated from enzymatically dissociated spleen using Lymphoprep density gradient (Accurate Chemical & Scientific Corporation, Westbury, NY). For cytokine intracellular staining (ICS), cells were stimulated for 4.5 hours with phorbol 12-myristate 13-acetate (PMA) (30 nM), ionomycin (1 μM) in presence of monensin (GolgiStop, BD Biosciences, San Jose, CA). Antibodies used for staining are listed in Supplementary Table S1. A live/dead (L/D) dye (Life Technologies, Carlsbad, CA) was used to exclude dead cells. FACS data were acquired using a LSRII cytometer (BD Biosciences) and analyzed with DIVA software (BD Biosciences). In some experiments, populations were cell sorted using Aria II (BD Bioscience). To block IL-17, mice were injected intra-peritoneally with 150ug of anti-IL17a mAb (Rat IgG1κ, clone TC11-18H10.1, Biolegend, San Diego, CA) or isotype control mAb twice weekly as described in each figure.