Intesticult medium

Small intestinal organoids were isolated and cultured as previously described^{55 (link)}. In brief, the small intestine was opened longitudinally and then washed in cold PBS with vigorous shaking, and the intestine was then cut into 2 mm pieces. The intestine samples were continuously washed in cold PBS with a pipette until the supernatant was clear. The samples were then incubated in 30 mM EDTA for 15 min at 4 °C and vortexed for 15 s to separate the crypts. Then the samples were filtered through a 70 μm cell strainer, followed by 200 × g centrifugation for 5 min. After being washed in cold PBS, the crypts were collected in a 50 ml centrifuge tube. For ISC isolation, the crypts were digested in 0.1% type I collagenase (Invitrogen) and incubated in 1× TrypLE Express (Life Technologies) supplemented with 1 kU mL⁻¹ DNase I (Worthington, USA) for single-cell preparation. Lgr5-GFP + ISC were classified using fluorescence-activated cell sorting (FACS) and incubated with IntestiCult medium (STEMCELL Technologies, Vancouver, Canada) for growing into organoids. To induce the organoid H/R model, organoids were placed in an anaerobic environment containing 5% CO₂, 2% O₂, and 93% N₂ in a 37 °C incubator for 4 h. Flow cytometry was performed using FACS Calibur instruments (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo (Tree Star Inc.).

Small intestinal enteroids were derived as previously described (Sato et al., 2009 (link)). In brief, small intestinal epithelial crypt fractions were isolated using 2 mM EDTA. A total of five epithelial fractions were isolated per genotype and crypt number, and purity was assessed through light microscopy. A total of 200-500 crypts were plated in 40 μl Matrigel (Corning) added to a 24-well dish. Cells were incubated in IntestiCult medium (StemCell Technologies) until harvesting for sphingolipid concentration determination.

All strains used in this study had a Salmonella enterica serovar Typhimurium SL1344 background (SB300; streptomycin resistant) (74 (link)). Besides the wild type (Salmonella Typhimurium WT), the previously described ΔinvG (64 (link)) and ΔsipA ΔsopBEE2 (referred to here as Salmonella Typhimurium Δ4) mutants (66 (link)) were used. The ΔmotA mutant was generated via transfer of a previously described deletion (75 (link)) from a Salmonella Typhimurium 14028 strain (C1172) to the SL1344 background by P22 transduction. Chloramphenicol-resistant, isogenically tagged Salmonella Typhimurium WT (tags A to C) and Salmonella Typhimurium ΔinvG (tags D to F) strains were used in an earlier study (65 (link)). The pFPV-mCherry (rpsM-mCherry; Addgene plasmid number 20956) (60 (link)), pM975 (pssaG-GFPmut2) (15 (link), 22 (link)), and pZ1400 (puhpT-GFP) (18 (link)) reporter plasmids were previously used and validated. For infections, Salmonella Typhimurium cultures were grown overnight for 12 h in LB–0.3 M NaCl (Sigma-Aldrich) with appropriate antibiotics, followed by subculturing in the same medium without antibiotics at a 1:20 dilution for 4 h. Prior to microinjection, the inoculum was reconstituted in antibiotic-free complete human or mouse IntestiCult medium (StemCell) at a concentration of 5 × 10⁸ to 1 × 10⁹ CFU/ml.

Intestinal crypts were isolated from the proximal part of the small intestine of 6–10 weeks Mask1 fl/fl Mask2 fl/fl mice (generated from EUCOMM ES cells for this study) as follows. The whole gut was harvested and washed in cold DPBS (#14190250, Gibco). The most proximal 5 cm were cut open, and the villi scraped off with a coverslip. The remaining tissue was cut in 0.5 cm pieces, washed several times by pipetting up and down in 8 mL fresh DPBS, and incubated in DPBS + 2 mM EDTA for 30 min at 4°C. Crypts were then mechanically extracted by vigorous shaking in DPBS, and filtered through a 70 µM Nylon cell strainer (#352350, Falcon). After several low speed washes in ADF-12 (#12634–010, Gibco), isolated crypts were resuspended and plated in Matrigel (#354230, Corning). Organoids were cultured in IntestiCult medium (#06005, Stemcell technologies) supplemented with Primocin antibiotic (1:50, ant-pm-2, Invivogen), either in 24-well plates for maintenance, or in 8-well chambers (#80827, Ibidi) for Ad-Cre-GFP infection and immunostaining. Passages were performed by resuspending Matrigel-embedded organoids in cold DPBS and transferring them to a Falcon tube using a 2 mL syringe with a 27 G ½ needle (BD Microlance #300635) to break them up. After two low speed washes in ADF-12, organoids were resuspended and plated in Matrigel.