1. Cell-culture medium: Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4-mM l-glutamine and 10% (v/v) fetal calf serum (Imperial Laboratories Europe, Winchester Hants, UK). Media may be optionally supplemented with the antibiotics penicillin (Sigma, Poole, Dorset, UK) at a concentration of 100 U/mL, and streptomycin (Gibco-BRL, Paisley, Scotland) at a concentration of 50 pg/mL. Store at 4°C.
2. DNA for transfection: 10 ||g at a concentration of 0.5-1 ||g/|L in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Plasmid DNA should be in the closed, circular form purified by CsCl-ethidium bromide density gradient centrifugation (9). Store at 4°C.
3. 2X HEPES-buffered saline (2X HBS): 280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, 12 mM dextrose, 50 mM HEPES, pH 7.05, with NaOH. Sterilize by filtration through a 0.22 |im filter. Store in 5-mL aliquots at -20°C.
4.2 M CaCl2: Sterilize by filtration through a 0.22-|im filter. Store in 5-mL aliquots at -20°C.
5. Phosphate-buffered saline (PBS): 0.138 MNaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.2. Sterilize by autoclaving. Store at 4°C or room temperature.
6. Wash solution: 0.02% (v/v) NP-40 in PBS. Store at 4°C.
7. Cell fixative: 0.2% (w/v) glutaraldehyde in wash solution. Make fresh from a 25% (w/v) solution (Sigma).
8. X-gal stain solution: PBS (pH 7.3) containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)63H2O, 2 mM MgCl2, 0.02% (v/v) NP-40, 0.5 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-ß-d-galactoside, Sigma). Store at 4°C in the dark. X-gal can be made up as a 100X stock solution (50 mg/mL in N,N-dimethylformamide— store in dark at -20°C). K3Fe(CN)6 and K4Fe(CN)63H2O can be made up as 0.5 M (100X) stock solutions and stored at 4°C in the dark.
9. Alkaline phosphatase (AP) stain solution: 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl2, 1 mg/mL NBT (nitro blue tetrazolium, Boehringer, Lewes, East Sussex, UK), 0.1 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), X-Phos, (Boehringer). Make up freshly. NBT can be made as a 50X stock solution (50 mg/mL in 70% [v/v] N,N-dimethylformamide—store in dark at -20°C). BCIP can be made as a 100X stock solution (10 mg/mL in distilled water—store in the dark at -20°C).
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