1. Gelatin-coated plates can be prepared in advance, stored in a clean place at room temperature or in a 37°C incubator.

2. The freezing medium should be kept at 4-8°C for no more than 5 d. It is recommended to use high-quality serum, such as FBS from Hyclone.

3. All types of trypsin-EDTA will do.

4. FBS or heat inactivated newborn calf serum is also suitable. It should be kept at 4-8°C and should be used within 2 wk of preparation.

5. 0.5% pronase may be used instead of the Tyrode's solution.

6. There may be differences between batches; therefore, it is recommended to prepare a 10X stock solution and dilute to the appropriate working concentration before use.

7. The guinea pig complement may become toxic if stored longer than 8 mo.

8. All culture media but those mentioned should be kept at 4-8°C for no more than 2 wk.

9. Additional suitable mice strains are CD1 and C56J6. The time frame of 12-14 d of conception is acceptable.

10. Any other sacrifice method approved by your animal care committee including the use of Avertin can be used.

11. The centrifugation step is optional; the serum-containing medium can be used to neutralize the Trypsin.

12. We recommend a ratio of three embryos per flask.

13. Adding the freezing medium drop by drop is crucial for cell recovery.

14. It is recommended to freeze four vials from one confluent flask.

15. The cells can be counted and frozen at a recommended volume of 106 cells per vial.

16. The use of Nalgene freezing box increases the survivability rates.

17. Adding the medium drop by drop is crucial for cell recovery.

18. The MEFs can be mitotically inactivated by exposure to 3500 Ci gamma irradiation; the duration of the exposure should be calculated according to the source in use.

19. We recommend on 4 x 105 cells per well in six-well plates. The MEF number can also be calculated as 4 x 104 cells/cm2. MEFs numbers can vary between 1.5 x 104 and 5 x 104 cells/cm2.

20. It is recommended to monitor the procedure; if trophoblasts are lysed before the end of the incubation time, stop the incubation.

21. It is not recommended to leave the vials at -80°C for less than 24 h or more than a few days.

22. The recommended freezing ratio is 10 cm2 per vial (one well in six-well plates).

23. For the cells to maintain their features, hESCs can be cultured and derived successfully using medium supplemented with either serum or serum replacement (for additional information, see ref. 30).


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