Gibco™ DMEM, high glucose, GlutaMAX™ Supplement

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Change media every 3-4 days

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Change media every 3-4 days

Publication protocol

Preparation of Organoids: The protocol was based on Mahe et al. [18]. Mice were sacrificed by CO2 inhalation followed by cervical dislocation at an age of 10-14 weeks. The entire colon was flushed and washed with sterile, ice-cold Dulbecco's phosphate-buffered saline (DPBS) in a sterile environment. All further steps were performed on ice. Tissues were opened lengthwise and cut into small pieces, washed several times with DPBS, transferred into crypt chelating buffer (CCB) (DPBS containing 0.5 M EDTA), and incubated for 30 minutes on a rocking platform. After washing with CCB, colon pieces were transferred into dissociation buffer (DB) (DPBS containing 54.9 mM sorbitol and 43.4 mM sucrose) and mixed thoroughly. Cell suspensions were filtered and centrifuged, and cells were resuspended in an appropriate volume of Matrigel® membrane matrix (Corning™, New York, USA) (Matrigel®) to yield approximately 1000 crypts/ml. Fifty μl of the cell-Matrigel® mixture was carefully pipetted into the centre of each well of a 24-well plate, incubated for 30 minutes at 37°C for solidification, and overlaid with 500 μl organoid growth media (DMEM, high glucose, GlutaMAX™, pyruvate (Thermo Fisher Scientific, Massachusetts, USA)) supplemented with 50% L-WRN-supernatant (ATCC® CRL3276™ in DMEM, high glucose, GlutaMAX™, pyruvate plus 10% fetal calf serum), 1x N2 (Invitrogen, Carlsbad, USA), 1x B27 (Invitrogen, Carlsbad, USA), 50 ng/μl recombinant mouse epidermal growth factor (Sigma-Aldrich, St. Louis, USA), 10 μM Y-27632 (Tocris, Bristol, UK), and 1x Cellshield (Biochrom, Berlin, Germany). Organoids were cultured at 37°C with 5% CO2. Media was exchanged every 3-4 days.

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Manufacturer protocol

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