40 μm filter

Small intestines were collected and processed as previously described to obtain lamina propria cells [84 (link)]. Briefly, small intestines were washed, then fat tissue and Peyer’s patches were removed. The cleaned tissue was cut longitudinally and trimmed into 1 cm pieces. The small intestine pieces were incubated twice for 20 minutes at 37°C in calcium/magnesium-free Hank’s Balanced Salt Solution (HBSS), 10 mM EDTA (VWR, Radnor, PA, USA), and 1 mM dithiothreitol (Sigma Aldrich, St. Louis, MO, USA) to release intraepithelial lymphocytes. A subsequent incubation for one hour at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) containing 300 U/mL collagenase (Worthington Biochemical, Lakewood, NJ, USA) was performed to release lamina propria cells. To purify the lamina propria cell population, cells were centrifuged without terminal braking in a discontinuous Percoll (Sigma Aldrich) gradient (40%/80% diluted with DMEM). The cells at the interface were collected and used for downstream applications. Mesenteric lymph nodes were collected, homogenized to release leukocytes, and passed through a 40 μm filter (VWR) prior to use in downstream applications.

For each tissue preparation, two age- and sex-matched mice were killed by live decapitation, and cervical through lumbar DRG were removed and pooled together. DRG were incubated in papain (45 U, Worthington) for 20 min at 37°C, 5% CO₂. DRG were then rinsed and incubated in collagenase (1.5 mg/mL, Sigma-Aldrich) for 20 min. Both enzyme solutions were made up in Ca²⁺- and Mg²⁺-free Hanks’ buffered salt solution (Corning) with 10 mm Hepes (Sigma-Aldrich). DRG were manually triturated with fire-polished Pasteur pipettes (VWR) to dissociate neurons, passed through a 40-μm filter (VWR), and plated onto poly-d-lysine/collagen (Sigma-Aldrich)-coated 12-mm glass coverslips (Thermo Fisher Scientific). Neurons were maintained in culture for 2 d in Neurobasal A medium (Invitrogen) supplemented with 100 U/mL penicillin/streptomycin (Corning), 2 mm GlutaMAX (Life Technologies), 2% B27 (Gibco), and 5% fetal bovine serum (Gibco).