Trichuris muris Infection and Tissue Preparation
In-house generated T. muris eggs were used for infection (13, 31). Experimental mice were infected with 200 T. muris eggs by oral gavage and euthanized at days 7, 14, 19, and 35 p.i. for analysis. At day 14, 19 and 35 p.i., the ceca from infected animals were collected to assess worm burden microscopically. Blood was collected for serum analysis. RNA tissue from the proximal colon was snap-frozen to analyze gene expression. Spleen, mesenteric lymph node (MLN) and cecum were collected to assess basophil infiltration using flow cytometry. Spleens and MLNs were digested with 1 U/mL Liberase TL (Roche) and 20 μg/mL DNase (Sigma-Aldrich) for 15 min at 37°C. Single-cell suspensions were prepared by disrupting the tissue through a 70-μm strainer. Spleens were lysed for red blood cells with ammonium-chloride-potassium lysis buffer (Lonza). For analysis of cecum basophil/immune cell populations, ceca were collected directly into ice-cold PBS, cut open longitudinally, cleaned of fecal contents, and washed twice in ice-cold HBSS (Thermo Fisher Scientific) with 10% FBS (Denville Scientific). Tissues were chopped roughly into 1-cm pieces in HBSS 2% FBS, stored on ice, and then shaken by hand vigorously for 10 s before being strained and incubated in HBSS supplemented with 2 mM EDTA (Thermo Fisher Scientific) for 15 min at 37°C in a shaking incubator. Samples were shaken by hand again, strained, and incubated for 25 min in fresh HBSS/EDTA at 37°C in a shaking incubator. After further shaking, samples were incubated in 1 U/mL Liberase TL and 20 μg/ml DNase for 35 min at 37°C in a shaking incubator with further vigorous manual shaking every 7 min. Samples were washed through 40-μm strainers to collect single-cell suspensions.