Preparation of Metaphase Chromosomes

Metaphase chromosomes were freshly prepared by using a spindle inhibitor such as Colcemid to arrest cultured cells during mitosis (see Notes 2 and 3).

1. Incubate 5 mL of cells suspension with 0.6 |g/mL Colcemid for 3 h at 37°C and 5% CO2.

2. Remove the cell suspension containing the Colcemid from the incubator and mix gently. Centrifuge for 6 min at 150g.

3. Remove the supernatant with a Pasteur pipet.

4. Add 1 mL of warmed hypotonic solution to the tube (see Note 2).

5. Mix gently.

6. Add 9 mL of hypotonic solution (see Note 10).

8. Centrifuge for 6 min at 150^.

9. Remove the supernatant.

10. Add drop by drop 5 mL of ice-cold fixative solution to the centrifuge tube (see Note 11).

11. Pipet with a Pasteur pipet to resuspend the pellet and mix the fixative.

12. Incubate this suspension at -20°C for 30 min (see Note 12).

13. Centrifuge for 6 min at 150^.

14. Remove the supernatant.

15. Add 6 mL of cold fixative and mix to resuspend the pellet. Centrifuge for 6 min at 150#.

16. Repeat steps 14 and 15.

17. Finally, the chromosome suspension is spread onto cleaned slide. Withdraw the cell suspension into a Pasteur pipet. From a height of approx 30 cm, drop two or three drops of fluid onto a clean slide.

18. Allow the slides to dry thoroughly (37°C, overnight).

19. Chromosomes are dehydrated in an ethanol series (70%, 90%, and 100%) for 2 min per step and are air-dried.

20. Slides can then be stored at room temperature until use in PRINS and FISH reactions.

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