Gibco™ DMEM/F-12, GlutaMAX™ supplement

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Growth of different organoid structures according to the supplements added to the medium

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Growth of different organoid structures according to the supplements added to the medium

Publication protocol

Isolation of murine colon and small intestinal crypts: Detailed procedures to isolate normal murine colon crypts have been described previously [29]. In brief, the colons were sterilized in 0.04% (w/v) sodium hypochlorite solution for 10 minutes at room temperature, followed by crypt isolation buffer (2mM EDTA/0.5 mM DTT in PBS) in a 50ml tube for 40 minutes in a 37°C water bath. The colons were transferred to PBS and the crypts were released into the PBS by manual shaking and pelleted at 100 x g for 1 minute at 4°C. Similarly, the small intestines were incubated in crypt isolation buffer for 5 minutes at 37°C. The small intestines were transferred into PBS and the villi were released by manual shaking 20 times (discarded). The small intestinal fragments were then transferred and incubated in fresh crypt isolation buffer for another 20 minutes at room temperature. The small intestines were again transferred into PBS and crypts were released by manual shaking 20 times. This was repeated for 3 times, with the first fraction discarded and the second and the third fractions pooled and pelleted at 100 x g for 1 minute at 4°C.
For the co-cultures of WEHI-YH2 cells/colon crypts, WEHI-YH2 cells (4000 cells/well) were seeded in complete DMEM (Life Technologies Corporation #11995073) in 96-well plates and incubated overnight. The medium was aspirated and the cells were washed once with pre-warm Growth Medium-I [1X N2 supplement (Life Technologies Corporation #17502048) and 1X B27 supplement (Life Technologies Corporation #17504) in DMEM/F12 medium (Life Technologies Corporation #10565042) supplemented with penicillin-streptomycin solution (1% v/v)]. The crypt suspension (100 crypts/15 μl Matrigel) was added to each well on the top of the monolayer of WEHI-YH2 cells. The Matrigel (BD Biosciences #356231) was allowed to polymerize at 37°C for 30 minutes. 100 μl Growth Medium-II [Growth Medium-I with 50 ng/ml recombinant mouse EGF (Peprotech #315–09), 100 ng/ml recombinant human noggin (Peprotech #120–10), 10 μM Y-27632 (Sigma-Aldrich #Y0503), human R-spondin 2-Fc conditioned medium (1% v/v) and Wnt3a conditioned medium (50% v/v) or partially purified Wnt3a conditioned medium (1% v/v)] was added to each well. Fresh Growth Medium-II was replaced every two days. After day 4, Y-27632 was omitted from the cocktail. For colonoid cultures using WEHI-YH2 conditioned medium, 2X Growth Medium-II was prepared and mixed with 0 to 50% (v/v) of WEHI-YH2 conditioned medium. Recombinant human Bone Morphogenetic Protein 4 (BMP4) (Gibco #PHC9534), TGF-β RI kinase inhibitor VI A83-01 (Tocris #2939) or EGFR tyrosine kinase inhibitor Tyrphostin AG1478 mesylate (Institute of Drug Technology (IDT) Australia, Batch DA68001) were added to the cultures as indicated in the individual experiments. Enteroid cultures were maintained under the same conditions as colonoid cultures except Wnt3a was not added. For some experiments (i.e. EGF perturbation and enteroid cultures) where the cultures were grown in 384-well plates (Corning™ #3707), ~40 crypts were embedded in 6 μl Matrigel were seeded in 384-well plates and 40 μl of Growth Medium II was added and changed every 2 days. R-spondin-Fc Conditioned Medium was harvested from 293F cells (after 7 days) transiently transfected with a human R-spondin2 construct that has Fc-fusion protein fused to the C-terminus and cloned into pApex vector.

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 Murine colonic organoids using Gibco™ DMEM/F-12, GlutaMAX™ supplement from Thermo Fisher Scientific.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing 3D Cell Culture Media Murine colonic organoids using Gibco™ DMEM/F-12, GlutaMAX™ supplement 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.