Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	After 2 days in culture remove CHIR and Rock inhibitor

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
After 2 days in culture remove CHIR and Rock inhibitor

Publication protocol

Isolation of small intestine (SI) and colon crypts from wild type mice was adapted from Sato et al. [66]. Briefly, resected prSI, diSI and colon were opened longitudinally, villi were removed and cut into 0.5 mm pieces. The fragments were washed several times with cold PBS. After this, the intestinal pieces were incubated in 2 mM (SI) or 25 mM (colon) EDTA in PBS at 4 °C for 30 min, in order to detach intestinal crypts from the basal layer. After removal of EDTA, crypts were resuspended in 10 mL of cold PBS 10% FCS, pipetted up and down vigorously, and passed through a 70 μm strainer. Subsequently, crypts were resuspended in Matrigel® (Corning), seeded in 24-well plates, and supplemented with Advanced DMEM/F12 medium (Gibco) containing 1× N2 (Gibco), 1× B27 (Gibco) 50 ng/mL, 10 mM HEPES (Boehringer), Glutamax (Gibco) and 1 mM N-acetylcysteine (Sigma-Aldrich), supplemented with 50 ng/mL murine EGF (TEBU-Bio) and 10% of Noggin and R-spondin1 conditioned media (hereafter called ENR medium). In addition, 10 μM CHIR (Axon Medchem) and 10 μM Rock inhibitor (Sigma-Aldrich) were added to all crypt cultures. After two days in culture, CHIR was removed from SI crypt cultures and Rock inhibitor from both, SI and colon. Cultures were incubated at 37 °C and 5% CO2.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing 3D Cell Culture Media Mouse small intestinal organoids using Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Gibco™Advanced DMEM/F-12 below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Mouse small intestinal organoids using Gibco™Advanced DMEM/F-12 from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.