Materials

1. PBS with Ca2+ and Mg2+. Prepare PBS-A by dissolving 10 g NaCl (Sigma S5885), 0.25 g KCl (Sigma P5405), 1.44 g Na2HPO4 (Sigma S5136), 0.25 g KH2PO4

(Sigma P5655) in 1 L of water. Autoclave. To 160 mL of PBS-A, add 20 mL of autoclaved 6.8 mM CaCl22H2O (Sigma C7902) and 20 mL of autoclaved MgCl2 6H2O (Sigma M2393).

2. Fetal calf serum (FCS) (Sigma, Ghent, Belgium).

3. Glycerol (Life Technologies, cat. no. 5514VA).

4. Freezing solution 1: PBS with Ca2+ and Mg2+ containing 10% (v/v) FCS.

5. Freezing solution 2: PBS with Ca2+ and Mg2+ containing 10% (v/v) FCS and 5% (v/v) glycerol.

6. Freezing solution 3: PBS with Ca2+ and Mg2+ containing 10%, (v/v) FCS and 10% (v/v) glycerol.

7. Freezing solution 4: PBS with Ca2+ and Mg2+ containing 10% (v/v) FCS 1, % (v/v) glycerol, and 0.6 M sucrose.

8. Sucrose (Sigma S1888).

9. M16 culture medium.

10. Freezing straws and plugs (Biotechnologies international, cat. no. ZA475 and ZA511, respectively).

11. Forceps.

12. Programmable freezing machine (e.g., Kryo 10 series II controlled-rate freezer from Planer Products Ltd., Sunbury, UK).

13. Liquid nitrogen storage tank.

14. 37°C incubator gassed with 5% CO2.

15. Stereomicrosope.

16. 35-mm Tissue-culture dishes.

17. Egg-transfer pipets and mouthpiece.

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