Intestinal Enteroid Generation and TNF-α-Induced Cell Death

Enteroids were generated from isolated small intestinal crypts of β-actin cKO and control mice, as previously described (Lechuga et al., 2017 (link)). Briefly, mice were euthanized and their small intestinal segments were dissected, longitudinally opened, and washed with ice-cold PBS. Crypts were released using 30 min incubation with PBS containing 2 mM of EDTA at 4°C, with constant agitation, followed by mechanical shaking. Debris and villous fragments were discarded, and the resulting crypt fraction was collected by centrifugation and resuspended in growth factor reduced Matrigel (BD Bioscience). After Matrigel polymerization, DMEM/F12 medium containing HEPES, glutamine, N2 and B27 supplements, and growth factors [50 ng/ml epidermal growth factor, 500 ng/ml R-spondin 1, and 100 ng/ml Noggin (R&D Systems)] were added. Intestinal enteroids were allowed to differentiate for 7 days and were observed using a bright field microscope (Olympus BX41, Japan). Cell death was induced by treating enteroids with 100 ng/ml of murine tumor necrosis factor (TNF)-α (PeproTech, Cranbury, NJ) for 12 h. Viable and dead enteroids were distinguished by morphology, using a bright-field microscope (Keyence, Osaka, Japan) and counted. At least 50 enteroids per experimental group were examined. The percentage of dead enteroids was calculated from 3 independent experiments.