Culturing Mouse Embryonic Stem Cells

The JM8.N4 cell line, derived from the mouse strain C57BL/6N [30 (link)], was cultured on 0.1% gelatin coated plates (Corning NY, USA) in Knockout DMEM (Gibco, UK) containing 2mM L-Glutamine (Gibco, UK), 0.05mM β-mercaptoethanol (Sigma Dorset, UK), 10% fetal bovine serum (Biosera East Sussex, UK) and 1000U/ml LIF (ESGRO, Millipore Watford, UK), at 37⁰ and 5% CO_2, changing medium daily. Cells are detached with 0.1% trypsin (Gibco, UK), containing 1% chicken serum (Gibco, UK), 0.6 mM EDTA (Sigma Dorset, UK) and 0.1% D-glucose (Sigma Dorset, UK) and passaged every two to three days. The E14 mESC line (129P2 background) was cultured in the same way but using GMEM with the addition of pyruvate and keeping the cells in 7% CO_2.