Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the BME in advance on ice or in fridge	Upon seeding or passaging, refresh medium every 3–4 d. Passage of organoids 7 d in an approximate ratio of 1 to 4 at a density of 2,500 viable cells per 10-µL BME drop

Upstream tips
Defrost the BME in advance on ice or in fridge
Protocol tips
Upon seeding or passaging, refresh medium every 3–4 d. Passage of organoids 7 d in an approximate ratio of 1 to 4 at a density of 2,500 viable cells per 10-µL BME drop

Publication protocol

Murine Epidermal Keratinocyte Isolation and Culture. Epidermal keratinocytes were isolated from telogen back skin collected from 8- to 18-wk-old mice, as previously described (28, 44). Viable keratinocytes were plated at a density of 2,500 cells per 10 µL BME (Cultrex) drop. Cells were cultured in EEM consisting of advanced DMEM/F12 (Gibco) supplemented with penicillin/streptomycin (100 U/L; Gibco), Hepes, (10 mM; Gibco), GlutaMAX (1×; Gibco), B27 supplement (50× stock; Gibco), N-Acetylcysteine-1 (1 mM; Sigma-Aldrich), Noggin-conditioned medium (5%, in-house), R-spondin 1-conditioned medium (5%, in-house, derived from 293T-HA-RspoI-Fc cells provided by Calvin Kuo, Stanford University, Stanford, CA), acidic FGF1 (100 µg/mL; Peprotech), Heparin (0.2%; Stemcell Technologies), Forskolin (10 ng/mL; Tocris), Rho kinase inhibitor (Y-27632; 10 µM; Sigma-Aldrich), and Primocin (500× stock; Invivogen). Additional components tested include FGF10 (100 µg/mL; Peprotech), TGF-β inhibitor (A-83-01; 2 µM; Tocris), and EGF (50 ng/mL; Invitrogen). Upon seeding or passaging, medium was refreshed every 3–4 d. Organoid cultures were passaged every 7 d in an approximate ratio of 1 to 4 at a density of 2,500 viable cells per 10-µL BME drop. Organoid cultures were stored long term in −80 °C or in liquid nitrogen. Two-dimensional feeder layer-dependent culture of murine epidermal keratinocytes was described elsewhere (28). Further details are given in SI Appendix, SI Materials and Methods.

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 skin 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 skin 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.