Isolation of Lamina Propria Mononuclear Cells

LPMCs were isolated from the SI specimens using a modification of a previously described protocol21 (link)59 . Briefly, the dissected mucosal tissue was incubated in calcium and magnesium-free Hank’s balanced salt solution (HBSS) (Life technologies, Carlsbad, CA) containing 2.5% heat-inactivated fetal bovine serum (FBS) (Life technologies) and 1 mM dithiothreitol (Sigma-Aldrich) to remove mucus. The mucosa was then incubated with agitation in HBSS containing 2 mM and 1 mM EDTA (Quality Biological, Gaithersburg, MD) for 15 min and 45 min, respectively, at 37 °C. 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.