Recombinant murine noggin

Small intestinal enteroids were generated from 4 to 8 week old wild type C57BL6 mice as previously described [51 (link)]. Enteroids were cultured in Advanced DMEM/F12 (Life Technologies 12,634) supplemented with 1x N-2 supplement (R&D Systems AR009), 1x B27 supplement (Gibco 17,504), 10 mM HEPES (Thermo Fisher Scientific15630080), 1x Glutamax (Gibco 15,630,080), 100 U/mL Penicillin-Streptomycin (Gibco 10,378,016), 50 ng/mL recombinant mouse EGF (Peprotech 315–09), 50 ng/mL recombinant murine noggin (Peprotech 250–38), and 50 ng/mL recombinant mouse r-spondin CHO-expressed (R&D Systems 7150-RS). Organoid cultures were passaged a minimum of one time prior to experimentation.

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.

Small intestine crypts were isolated from the small intestine of 4–8-week-old C57BL/6 mice and embedded in a 50% Matrigel (Corning 352231) solution in complete enteroid media consisting of 50 ng/ml recombinant mouse EGF (Peprotech 315-09), 50 ng/ml recombinant murine noggin (Peprotech 250-38), and 50 ng/ml recombinant mouse r-spondin (R&D Systems 7150-RS) as previously described^{80 (link)}. Enteroids were passaged a minimum of one time prior to experimentation in 24-well plates at approximately 150 organoids per well. For whole mount immunofluorescent staining, samples were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.1% Triton X-100 for 30 min at room temperature, then blocked in an ice-cold PBS solution containing 5% normal goat serum and 1% BSA for 60 min. Fixed organoids were then incubated overnight at 4 °C with DCLK1 antibody (Abcam, Cat# ab31704, 1:400 dilution) and followed by overnight at 4 °C with Alexa Fluor 488 goat anti-rabbit secondary antibodies (Thermal Fisher, Cat# A11008, 1:2000 dilution). Samples were subsequently counterstained with a 1:5000 DAPI solution (Fisher Scientific 62248) for 1 h at room temperature and stored at 4 °C until visualization using a confocal microscope (Zeiss 710 confocal microscope).