Serum-Free Culture of Mouse Embryonic Stem Cells

Mouse ESCs E14 were grown in culture dishes coated with 0.1% gelatin (Sigma) in Glasgow Minimum Essential Medium (GMEM, Gibco) supplemented with 15% fetal bovine serum (FBS, AusGeneX), 100 nM nonessential amino acids (Gibco), 1% sodium pyruvate (Gibco), 200 mM glutamate (Gibco), 1% penicillin-streptomycin (Gibco), 50 μM β-mercaptoethanol (Sigma), 10 ng/ml LIF (ESGRO), 1 μM PD0325901 (a MEK inhibitor, Selleckchem) and 3 μM CHIR99021 (a GSK inhibitor, Selleckchem). The medium was replaced every 2–3 days (32 (link),33 (link)). Cells are maintained at 37 °C in a 5% CO₂ incubator.

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Su G., Guo D., Chen J., Liu M., Zheng J., Wang W., Zhao X., Yin Q., Zhang L., Zhao Z., Shi J, & Lu W. (2019). A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation. Nucleic Acids Research, 47(13), 6737-6752.

Publication 2019

gelatin nonessential amino acids sodium pyruvate glutamate penicillin-streptomycin β-mercaptoethanol PD0325901 CHIR99021

Amino acids Cells Chir99021 Dishes Escs Gelatin Glutamate Gmem Mercaptoethanol Mouse Pd0325901 Penicillin Pyruvate Sodium Streptomycin

Corresponding Organization : Broad Center

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Variable analysis

independent variables

Presence of 0.1% gelatin (Sigma) coating on culture dishes
Concentration of fetal bovine serum (FBS, AusGeneX) at 15%
Concentration of nonessential amino acids (Gibco) at 100 nM
Concentration of sodium pyruvate (Gibco) at 1%
Concentration of glutamate (Gibco) at 200 mM
Concentration of penicillin-streptomycin (Gibco) at 1%
Concentration of β-mercaptoethanol (Sigma) at 50 μM
Concentration of LIF (ESGRO) at 10 ng/ml
Concentration of PD0325901 (a MEK inhibitor, Selleckchem) at 1 μM
Concentration of CHIR99021 (a GSK inhibitor, Selleckchem) at 3 μM

dependent variables

Growth and maintenance of Mouse ESCs E14 in culture

control variables

Culture temperature at 37 °C
CO2 concentration at 5%
Medium replacement frequency of 2-3 days

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