Maintenance of Mouse ES Cells

1. Cell culture medium (IX): store up to 1 mo at 4°C:

a. 10X Glasgow MEM/BHK12 (Gibco, Paisley, UK): store at 4°C 40 mL

b. 7.5% sodium bicarbonate (Gibco): store at 4°C 13.2 mL

c. 1X MEM nonessential amino acids (Gibco): store at 4°C 4 mL

d. 200 mM glutamine, 100 mM sodium pyruvate (Gibco):

e. 0.1 M 2-mercaptoethanol (Sigma, Dorset, UK): 0.4 mL store at 4°C for 1 mo f. Fetal calf serum (Globepharm, Surrey, UK), batch-tested

g. Sterile, deionized water (dH2O) 340 mL

h. Differentiation inhibiting activity (DIA) (see Note 1)

2. Phosphate-buffered saline (PBS), filter-sterilized.

3. Trypsin solution: Dissolve 250 mg of trypsin (Difco, Surrey, UK) and 372 mg EDTA disodium salt (Sigma) in 1 L of PBS. Add 10 mL of chicken serum (Flow Labs, Herts, UK and filter sterilize. Store in 20-mL aliquots at -20°C.

4. 1% (w/v) Gelatin: add 1 g gelatin (Type A from bovine skin, Sigma) to 100 mL dH2O, autoclave, and store in 20-mL aliquots at 40°C. For a working solution of 0.1%, add 10 mL of 1% gelatin to 90 mL of PBS.

5. 0.1 M 2-mercaptoethanol: Add 100 mL 2-mercaptoethanol (Sigma) to 14.1 mL PBS. Store up to 1 mo at 4°C.

6. Geneticin (G 418; Boehringer-Mannheim, Sussex, UK): 200 mg/mL dissolved in dH2O.

7. Freezing solution: 1 mL of DMSO to 9 mL of cell-culture medium (made fresh).

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