Intesticult organoid growth medium mouse

Pellet crypts were resuspended in Matrigel (Corning, New York, USA). Briefly, 50 μL of Matrigel-containing crypts was plated in the center of wells in a 24-well plate. After polymerization at 37 °C, 500 μL of the medium (IntestiCult Organoid Growth Medium (Mouse), STEMCELL Technologies, Vancouver, Canada) was added, and a protocol adapted from manufacturer’s instructions as previously reported was used [31 (link)]. The culture medium was changed three times per week. After 7–10 days, organoid cultures were passaged. The organoid medium was removed, and Matrigel including organoids was dissolved with Gentle Cell Dissociation Reagent (STEMCELL Technologies, Vancouver, BC, Canada). Following centrifugation at 200× g for 5 min, the supernatant was removed and pelleted organoids were resuspended in Matrigel. All experiments were performed at least after two passages.

Small intestinal and colon organoids were isolated from the mouse intestine and cultured for a minimum of 6 days as described earlier (Günther et al., 2011 (link)). In brief, crypts were isolated by incubating pieces of the small intestine or colon of 8‐ to 12‐week‐old C57BL/6J mice rolling in isolation buffer (Phosphate buffered saline (PBS), 2 mM EDTA; 4°C) and consequent repetitive steps of vortexing, washing (1xPBS) and centrifugation (5 min, 200×g). Finally, crypts were transferred in 25 μl Corning Matrigel into 48 well plates, solidified (10 min, 37°C) and then covered with 300 μl intestinal culture medium. Medium for small intestinal organoids contained [Advanced DMEM/F12 (Invitrogen), containing HEPES (10 mM, PAA), GlutaMax (2 mM, Invitrogen), Penicillin (100 U/ml, Gibco), Streptomycin (100 μg/ml, Gibco), B27 Supplement 1x (Invitrogen), 1 mM N‐acetylcysteine (Sigma‐Aldrich), murine EGF (50 ng/ml, Immunotools); R‐spondin and recombinant murine Noggin was added equivalent to recombinant human R‐spondin (1 μg/ml, R&D Systems), and recombinant murine Noggin (100 ng/ml, Peprotech). For colon organoids IntestiCult Organoid Growth Medium (Mouse) (STEMCELL Technologies) was used. To both intestinal organoid culture media extra WNT3a (0.25 nM, U‐Protein Express BV) was added to promote additional stem cell expansion.

Mouse crypts were isolated and cultured following previously described methods [17 (link),18 ]. Briefly, intestines were obtained from 8- to 10-week-old C57BL/6 mice, and the fecal matter was flushed with ice-cold DPBS (HyClone). After longitudinally cutting and washing the intestine with ice-cold DPBS, it was cut into 2–3 mm pieces and incubated in dissociation buffer (DPBS with 30 mM EDTA) for 20 min on ice. After replacing the dissociation buffer with DPBS, pieces of intestine were incubated in a 50 ml tube for another 15 min on ice. After the tube was shaken for 30–60 s, the mixture was passed through a 70 mm cell strainer to isolate and purify crypts via centrifugation at 4°C. Then, crypts mixed with Matrigel (Corning) were seeded in 96-well flat-bottom plates (10 ml/well). The plates were incubated at 37°C for 17 min. Culture medium, IntestiCult™ Organoid Growth Medium (Mouse) (Stem Cell Technologies, Cat: 06005) was added to the wells (100 ml/well). Growth factors [R-spondin 1, epidermal growth factor (EGF) and Noggin] were added every other day and the entire culture medium was replaced after 4 days of seeding. Y-27632(Sigma, Cat:Y0503-1MG) was then withdrawn after 4 days of seeding and not included in subsequent culture maintenance.

The small intestine was flushed with ice-cold phosphate buffered saline and cut into 1 cm segments and then transferred into BSS buffer (1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, 8 mM KH₂PO4, 5.6 mM Na₂HPO4 and 15 mM EDTA) (isolation buffer), vortexed at 4 °C for 10 minutes, transferred into fresh isolation buffer, and vortexed for another 10 minutes. The crypts and villi were separated via a 70 µm cell strainer, spun down at 500 g for 3 minutes and washed once with ice-cold PBS. For ex vivo stimulation, the enterocytes were resuspended in DMEM/F12 medium and stimulated with 50 ng/ml IL-4 (#5208, Cell Signaling Technology, Danvers MA) or IL-13 (#210-13, PEPROTECH, Rocky Hill NJ) in a tissue culture incubator for 10 minutes. The enterocytes were collected, lysed with sodium dodecyl sulfate sample buffer and analyzed by Western blot assay. For organoid culture, the crypt fraction was mixed with matrigel (Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, #356231, Corning NY) and the standard protocol^{17 (link)} was followed to generate organoids using IntestiCult™ Organoid Growth Medium (Mouse) (#06005, STEMCELL Technology, Cambridge MA). To induce tuft cell differentiation, the organoids were stimulated with 50 ng/ml IL-4 for at least 48 hours. The organoids were then fixed for immunofluorescence, or harvested for protein & mRNA analysis.

HKST was purchased from InvivoGen Asia (Hong Kong, China) and suspended in phosphate buffered saline (PBS). PBS and RMPI1640 medium were obtained from Hyclone (Logan, UT). ICG-001 was obtained from Selleck Chemicals (Shanghai, China). IntestiCult Organoid Growth Medium (Mouse) was purchased from STEMCELL Technologies Inc. (Canada). Growth factor-reduced Matrigel was purchased from Corning Incorporated (USA) and stored in −20°C.

For preparation of intestinal stem cells, 100 mM phosphate buffered saline (PBS) was used; for stem cell isolation 2 mM EDTA (ethylenediaminetetraacetic acid) was dissolved in PBS (EDTA/PBS). Organoid growth medium (IntestiCult™ Organoid Growth Medium Mouse) was obtained from StemCell Technologies (Vancouver, Canada).
Tyrode solution, which was used for Ca²⁺ imaging experiments, consisted of (in mM): 140 NaCl, 5.4 KCl, 10 HEPES [N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid], 12.2 glucose, 1.25 CaCl₂, and 1 MgCl₂. For Ca²⁺ imaging experiments with Ca²⁺-free Tyrode solution, CaCl₂ was omitted. The immunofluorescence experiments were carried out in phosphate buffer (PB) containing 80 mM Na₂HPO₄ and 20 mM NaH₂PO₄. For Tyrode solution and PB the pH was set to 7.4 with 1 N HCl/NaOH.