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.