Preparation of Media and Solutions

1. Medium: 10 mL 10X DMEM with NEAA without l-glutamine, sodium pyruvate, or NaHCO3 (Gibco-BRL, Paisley, Scotland, UK, no. 12501-011): 7 mL 5.3% NaHCO3, 1 mL penicillin/streptomycin, 58.4 mg l-gluamine (Sigma), 82 mL auto-claved milli-Q H2O. Filter-sterilize and adjust pH to 7.4 with 800 |L 1 N NaOH.

2. Medium with serum: 15 mL medium (see item 1), 3 mL heat-inactivated fetal bovine serum, 2 mL heat-inactivated horse serum.

3. Growth medium: Medium with serum, add 0.8 mg/mL G418 (Geneticin G-418 sulphate; Gibco-BRL no. 11811-01E), filter-sterilize.

4. Coated dishes: Make fresh a 0.1% gelatin solution (Sigma) in PBS by gentle heating and shaking. Filter-sterilize while still warm. Completely cover the base of tissue-culture dishes. Leave for 2 h at room temperature, and then aspirate off and wash 1X with sterile PBS. Plate cells or cover with fresh PBS and store at 4°C (maximum 2 wk).

5. Serum-free medium: 10 mL media, 20 |L N3 (serum supplement—see item 6).

6. N3 serum supplement: 246 |L Hank's balanced salt solution (HBSS) without calcium and magnesium, 50|L 10 mg/mL bovine serum albumin in HBSS (store at 4°C), 100 |L 100 mg/mL human transferrin in HBSS (store at -20°C), 20 |L 80 mg/mL putrescine hydrochloride in HBSS (store at -20°C), 50 |L 10 mM sodium selenate in HBSS (store at -20°C), 5 |L 20 mg/100 mL triodothyronine, sodium salt in 0.01 M NaOH (store at -20°C), 20 |L 25 mg/mL bovine pancreatic insulin in 20 mM HC1 (store in plastic at -20°C), 5 |L 12.5 mg progesterone in absolute alcohol (store in glass at -20°C), 1 |L 2 mg/mL corticosterone in absolute alcohol (store in glass at -20°C).

7. X-gal stain: 3.08 mL 0.5 MNa2PO4 0.46 mL 1 MNaH2PO4, 0.026 mL 1 MMgCl2 1.2 mL 50 mM K3Fe(CN)61.2 mL 50 mM K4Fe(CN)6, 1.0 mL X-gal stock = a 2% solution of 5 bromo-4 chloro-3-indoxyl-P-d galactopyranoside (Novabiochem) made up in N-N-dimethylformamide (Sigma), 13 mL milli-Q H2O.

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