Tissue Culture see Notes 1 and

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1. Knockout DMEM-optimized Dulbecco's modified Eagle's medium for ES cells (Gibco BRL, Carlsbad, CA; cat. no. 10829-018).

2. DMEM 4.5 g/L glucose (Sigma, Dorset, UK; cat. no. D5796).

4. Nonessential amino acids 100X stock (Biological Industries, Kibutz Beit-Haemek, Israel; cat. no. 01-340-1B).

5. Insulin-transferrin-selenium 100X (Gibco BRL; cat. no. 41400-045).

11. KnockOut SR—serum-free formulation (Gibco BRL; cat. no. 10828-028).

12. Fetal calf serum (Biological Industries).

13. l-glutamine 100X stock (200 mM/L, Biological Industries; cat. no. 03-020-1).

14. Penicillin (10,000 U/mL) and streptomycin (10 mg/mL) 100X stock (Biological Industries; cat. no. 03-031-1B).

15. Human basic fibroblast growth factor (bFGF) stock solution (2 ng/|L) (human recombinant; Gibco BRL; cat. no. 13256029).

16. Trypsin-EDTA: 0.25% trypsin and 0.05% EDTA (Biological Industries; cat. no. 03-052-1).

19. Dimethylsulfoxide (DMSO; Sigma; cat. no. D-2650).

20. 1X phosphate-buffered saline (PBS) without Ca2+/Mg2+. For 1 L: mix 3.58 g sodium phosphate (Na2HPO412H2O), 0.24 g potassium phosphate monobasic (KH2PO4), 8 g sodium chloride (NaCl), 0.2 g potassium chloride (KCl), in a final volume of 1 L of double-distilled water (dd^O). Aliquot 200 mL solution per bottle and autoclave, store at room temperature.

21. 10 mM ß-mercaptoethanol: dilute 1:100 in PBS, filter, sterilize, and store at 4°C.

22. 50X Mitomycin-C: dissolve 2 mg in 4 mL MEF medium, store in 4°C.

23. bFGF solution: add 10 |g of bFGF solution to 5 mL of filter-sterilized 0.1% bovine serum albumin dissolved in 1X PBS (with Ca2+/Mg2+), to give a final concentration of 2 |g/mL, store 1-mL aliquots in -20°C.

24. 0.1% gelatin solution: add 0.1 g of gelatin into a bottle containing 100-mL distilled water and autoclave immediately. The gelatin is dissolved while boiling in the autoclave, store at 4°C.

25. MEF media: add to a 500-mL bottle of DMEM (high glucose and l-glutamine) 50-mL fetal calf serum and 2.5 mL penicillin/streptomycin.

26. hES medium: add to a 500-mL bottle of Knockout DMEM: 75 mL KnockOut SR, 6 mL nonessential amino acids, 6 mL glutamine (2 mM), 3 mL insulin-transferrin-selenium, 60 |L ß-mercaptoethanol (0.1 mM), 3 mL penicillin/streptomycin, and 1 mL bFGF. ES media should be protected from light (see Note 3), and stored in 4°C up to 1 mo.

27 Freezing medium: add 1 mL of DMSO to 9 mL of appropriate media (either hES or MEF media). Media should be prepared fresh.

28. Leishman's stain (BDH, Poole, England) in 100% methanol.

2.1.1. Equipment and Supplies for Tissue Culture

1. Laminar flow hood.

2. Humidified incubator set at 37°C and 5% CO2.

3. Phase contrast microscope (objective range from x10 to x40).

4. Liquid nitrogen storage tank.

5. Refrigerator (4°C) and freezers (-20°C, -70°C).

7. Swing-out centrifuge for conical tubes (15- and 50-mL).

8. Cell counter.

9. Pipetmen (2, 10, 20, 200, and 1000 ||L) designated for tissue culture use only.

10. Sterile forceps and scissors for dissecting mouse embryos.

11. Falcon tissue culture plates (100 x 20 mm) and 6-, 12-, and 24-multiwell trays (Falcon, Bedford, MA; cat. no. 353047, 353047, 353043, 353046).

12. Falcon 15-mL and 50-mL (Falcon; cat. no. 352097, 352098) polypropylene conical tubes.

13. Cryo vials (1.8-mL CryTube; Nunc, Roskilde, Denmark; cat. no. 363401).

15. Tips for 2-, 10-, 20-, 200- and 1000-|L pipetmen.

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