Isolating Colonic Lamina Propria Cells

Colonic lamina propria cells were isolated, as previously described [18 (link)]. Briefly, colon tissues were washed in PBS containing 1 mM DL-dithiothreitol (DTT; Sigma-Aldrich, Irvine, UK), 30 mM ethylenediaminetetraacetic acid (EDTA; Thermo Fisher Scientific/Ambion, Waltham, MA, USA), and 10 mM 4-[2-hydroxyethyl]-1-piperazineerhanesulfonic acid (HEPES; Thermo Fisher, Waltham, MA, USA) at 37 °C for 10 min. The tissues were washed again in PBS containing 30 mM EDTA and 10 mM HEPS at 37 °C for 10 min. After washing, the tissues were digested in RPMI 1640 containing 0.5 mg/mL collagenase VIII (Sigma-Aldrich, Chem, Fort Lauderdale, FL, USA) and 15 ug/mL DNase I (Sigma Aldrich; 90 mg/mL) at 37 °C for 1 h. The cell suspensions from the enzyme digestion were then applied to a Percoll (GE Healthcared/Amersham, Bucking-hampshire, UK) gradient (for lymphocytes: 40% Percoll on the top and 80% Percoll on the bottom) by centrifugation without breaking at room temperature.