4 2 hydroxyethyl 1 piperazineethanesulfonic acid

LP extraction was performed according to Moreira’s protocol^{38 (link)} with few modifications. In brief, feces and mucus were removed from the collected intestines. Tissue was flipped (inside out) and washed several times using Hank’s Balanced salt solution (HBSS) (Gibco) (without MgCl_2, CaCl₂, and MgSO₄) solution supplemented with 5% FBS (Gibco) and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Lonza). After cleaning, the entire gut segment was incubated for 20 min at 37°C in an orbital shaker with HBSS supplemented with 15 mM HEPES (Sigma) + 5 mM ethylenediaminetetraacetic acid (EDTA) (Invitrogen) 10% FBS + 0.015% or 1 mM 1.4-dithiothreitol (DTT; Roche). After incubation, tissues were washed two times with HBSS-EDTA solution supplemented with 25 mM EDTA. Tissues were chopped into smaller pieces and incubated with exVIVO (Lonza) media containing Liberase TL (Roche) 5 mg/80 ml + Dnase 30 mg/ml, grade 1 (Sigma-Aldrich) for 45 min in a 37°C shaker. The supernatant containing LP cells was meshed using a 100 μm and 70 μm cell strainer and washed with HBSS solution supplemented with 2 mM EDTA 5 mM HEPES and 5% FCS. The cell suspension was then purified using CD45 microbeads according to the manufacturer’s protocol (Miltenyi Biotec). The sorted cell suspension was resuspended in FACS buffer and stained for flow cytometry.