1. Alpha minimum essential medium (a-MEM): for economy, we buyprepared medium as powder. A 44 g quantity of sodium bicarbonateis added, the powder is made up to 20 L in deionized distilled water,the pH adjusted to 7.4, and the media sterile-filtered through a 0.22 wfilter using a pressure vessel coupled to a filtration apparatus anddriven by a pressurized 95:5% CO, gas mix. This gas mix maintainspH upon storage. The 500 mL bottles are stored at 4OC n the dark untiluse (seeNotes 1 and 2).2. Growth medium: a-MEM plus 15 or 7.5% (v/v) fetal calf serum. Thisis made up as required and stored at 4OC.3. Ca2+Mg2+-free Phosphate Buffered Saline (Dulbecco, PBS): NaCl, 8 g/L; KCl, 0.2 g/L; KH,PO, 0.2 g /L; Na,HPO,
7HZ0, 2.31 g /L pH 7.2.4. PBS citrate: PBS + sodium citrate at 5.88 g/L.5. Trypsin: One vial of lyophilized Difco Bacto-trypsin in 400 mL of PBScitrate (0.125% trypsin) or 10 x this concentration for the isolation ofembryonic fibroblasts (seeNote 3).6. Counting fluid; PBS + 0.2% (v/v) FCS.7. Formalin fixative: 10% (v/v) commercial formaldehyde (comes as a40% v/v solution).8. Methylene blue stain: 0.1% (w/v> methylene blue in distilled waterfiltered through a Whatman No. 1 filter.9. Trypan blue: 0.5% (w/v) in PBS.10. Colcemid: 10 pg/mL, stored at 4OC.11. Karyotype fix: Methanol: acetic acid (3:l) made up on the day of useand kept on ice in a tightly stoppered bottle.12. Giemsa stain: Use commercial Giemsa concentrate diluted 3:47 partsin commercial Gurr’s buffer (one tablet to 1 L distilled water). Alter-natively, 10 mM potassium phosphate, pH 6.8, can be used as thebuffer. The diluted stain is only stable for 2-3 mo.
Establishment of Primary ChineseFibroblast CuZtures
3.1.1. Embryo Culture
1. Kill a 10-12-d pregnant Chinese hamster with ether.2. Wash the animal in tap water and then with 70% ethanol.