Mouse recombinant tnf α

BM cells from tibiae and femurs were collected from 7- to 8-wk-old mice. Red blood cell-free BM cells were incubated with α-Minimum Essential Medium (MEM) containing 10% FBS and penicillin/streptomycin for 3 h on Petri dishes. Non-adherent BM cells were seeded onto 6-well plates (5.0 × 10⁵ cells/well) and cultured with M-CSF (25 ng/mL, PeproTech) for 4 days to expand bone marrow-derived M-CSF-dependent macrophages (BMMs). Subsequently, BMMs were cultured without M-CSF for 4 h, then stimulated with E. coli LPS (Invivogen) or mouse recombinant TNF-α (PeproTech).

Mouse colon stem cells were cultured using IntestiCult Organoid Growth Media according to the manufacturer's instructions (06005, STEMCELL Technologies) [19 (link)]. Small intestines 20 cm in length nearly to the stomach were removed from untreated WT,MPST^+/- and MPST^−/− mice and rinsed with cold PBS. The colon was cut into 2 mm segments and washed 15 times with cold PBS. Colonic segments were incubated in Gentle Cell Dissociation Reagent (07174, STEMCELL Technologies) and rotated at 350 g for 15 min at room temperature, followed by resuspension in 0.1% BSA (A6003, Sigma) in PBS. Dissociated colonic crypts were filtered through 40μm strainers. Dissociated colonic crypts were resuspended in DMEM/F12 medium with 15 mM HEPES (36254, STEMCELL Technologies), counted and resuspended in IntestiCult Organoid Growth Media and Matrigel (356230, Corning) in a 1:1 ratio. Cells were plated in 24-well culture plates (3738, Corning). IntestiCult Organoid Growth Media was added to the cell culture plates to immerse the matrix composed of IntestiCult Organoid Growth Media and Matrigel. The culture medium was changed every other day, and the organoids were observed daily. After 5–7 days, the organoids were stimulated with 100 ng/mL mouse recombinant TNF-α (PeproTech, China) for 24 h.