Isolation of Lamina Propria Mononuclear Cells

Lamina propria mononuclear cells (LPMCs) were isolated from SI specimens using a modification of a previously described protocol^{5 (link),32 (link)}. Briefly, the dissected mucosal tissue was incubated in calcium and magnesium-free Hank’s balanced salt solution (HBSS) (Life Technologies, Carlsbad, CA) containing 1.5% heat-inactivated fetal bovine serum (FBS) (Life Technologies) and 1 mM dithiothreitol (Sigma-Aldrich) to remove mucus. Epithelial cells were removed by incubation in HBSS containing 1 mM EDTA (Quality Biological, Gaithersburg, MD) at 37 °C, repeated 2 times (first incubation; 10 min, second incubation; 30 min). The tissues were then collected and incubated, with agitation, in HBSS containing 400 U ml⁻¹ of type 3 collagenase and 0.01 mg ml⁻¹ of DNase I (Worthington Biochemical, Lakewood, NJ) for 90 min at 37 °C. The insoluble fraction was pelleted, re-suspended in a 40% Percoll solution (GE Healthcare Life Sciences, Pittsburgh, PA), layered on top of a 75% Percoll solution and centrifuged at 2,000 r.p.m. for 20 min at room temperature. Viable LPMCs were recovered from the discontinuous gradient interface.