ES-DMEM
DMEM (high-glucose, 4500 mg/L) can be purchased as a powder or ready-made (e.g., Invitrogen). If it is reconstituted, use water free of bacterial endotoxins. Before use, supplement with:
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Glutamine to 2 mM from a 100X stock (200 mM, e.g., Invitrogen), stored as aliquots at −20°C.
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MEM nonessential amino acids to 0.1 mM from a 100X stock (10 mM, e.g., Invitrogen), stored at 4°C.
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0.1 mM β-mercaptoethanol or 0.15 mM monothioglycerol (both tissue culture grade, Sigma), stored as aliquots at −20°C.
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(Optional) Sodium pyruvate to 1 mM from 100X stock (e.g., Invitrogen), stored at 4°C.
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Gentamicin to 50 μg/mL (50 mg/mL stock, Sigma), or penicillin to 50 U/mL and streptomycin to 50 μg/mL, or no antibiotics, particularly if cells are to be tested for Mycoplasma. A 100X stock of pen-strep (e.g., Invitrogen) is stored at −20°C.
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15% fetal bovine serum (FBS) (e.g., Invitrogen, Sigma). Identify a batch that gives optimal growth and reserve a supply if possible.
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Leukemia inhibitory factor (LIF), 500-1000 U/mL for MEF feeders and 1000 U/mL for STO feeders (ESGRO/Millipore).
Store in the dark at 4°C. If complete medium is stored for >2 wk, supplement with additional 1-2 mM L-glutamine.
