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#.
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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