http://www.jax.org/staff/mills/protocols/preparation_of_metaphase.pdf


Preparation of Metaphase Spread
Method I
1. Add colcemid (1:1000 KaryoMAX) directly to culture dish and swirl.
Incubate 30 min to 2 hours.
Metaphases can be prepared without colcemid. Colcemid should increase the
number of metaphase chomosomes but longer incubation times will result in shorter,
more compact chromosomes.
2. Trypsinize cells as normal and wash 1X 10ml PBS. At this point it is no longer
necessary to be sterile.
3. Remove as much PBS as possible and gently resuspend the cells in the
residual.
4. Slowly add 0.075M KCl dropwise to 10ml. I add 1-2 drops then invert
the tube. As soon as there are about 3 ml of KCl in the tube addition can
become faster.
5. Incubate at 37oC (in a water bath) for EXACTLY 6 minutes.
6. Centrifuge at 900rpm for 5 minutes.
7. Remove as much KCl as possible and gently resuspend the cells in the residual.
8. SLOWLY add 5 ml of fixative (3:1 Methanol/Acetic acid; prepared fresh)
dropwise and carefully mix the whole time. Adding fixative too quickly will
result in clumping.
9. Centrifuge at 900rpm for 5 minutes and remove fixative
10. Slowly add 2 ml fixative dropwise.
11. Centrifuge 900rpm 5 minutes and remove all but 200-500μl of the fixative.
Cells are stable for extended times in fixative. If desired, store at 4oC.
12. Drop a few drops from about 18 inches high onto angled, humidified
microscope slide.
13. IMMEDIATELY blow on the slide very gently.
14. Air dry at least 10 minutes. Slides are now stable for a long time.
Preparation of Metaphase Spread
Method II
1. Add colcemid (1:1000 KaryoMAX) directly to culture dish and swirl.
Incubate 30 min to 2 hours.
Metaphases can be prepared without colcemid. Colcemid should increase the
number of metaphase chomosomes but longer incubation times will result in shorter,
more compact chromosomes.
2. Trypsinize cells as normal and wash 1X 10ml PBS. At this point it is no longer
necessary to be sterile.
3. Remove as much PBS as possible and gently resuspend the cells in the
residual.
4. Slowly add KCl dropwise to 10ml. I add 1-2 drops then invert
the tube. As soon as there are about 3 ml of KCl in the tube addition can
become faster.
use 0.4% KCl for MEFs
use 0.57% KCl for lymphocytes, ES cells
5. Incubate at 37oC (in a water bath) for EXACTLY 8 minutes.
6. Centrifuge at 900rpm for 5 minutes.
7. Remove as much KCl as possible
8. Gently add 10 ml of cold fixative (3:1 methanol/acetic acid prepared fresh) and
gently but quickly resuspend the pellet by pipetting up and down.
9. Centrifuge at 900rpm for 5 minutes
10. Repeat steps 8 and 9
11. Remove all but 1-2 ml of fixative and gently resuspend pellet in remainder
12. Store at 4oC or proceed to dropping metaphases onto slides.
Preparation of Metaphase Spread
Method III
(cultured lymphocytes cells)
1. Add KaryoMax colcemid to culture at a 1:200 dilution. Incubate 3.5-4.5 hours.
2. Spin cells down in tabletop clinical centrifuge for 5 min at 1000rpm in 15ml
concial tubes.
3. Remove supernatant and gently resuspend in 1ml PBS pH 7.4 by pipetting up and
down with a P1000.
4. Spin cells down in microfuge for 2min at 1000 RCF.
5. Very gently remove most of the supernatant with a P1000, being careful not to
disturb the pellet. Gently resuspend in the residual PBS by flicking the tube or
pipetting up and down with a p200.
6. Add 900μl 0.075M KCl. Incubate at 37oC for 17 min.
7. Slowly and gently add 100μl cold, fresh fixative (3:1 methanol/acetic acid).
Invert to mix.
8. Spin down in a microfuge for 2min. at 1500 RCF.
9. Very gently remove most of the supernatant with a P1000, being careful not to
disturb the pellet. Gently resuspend in the residual fixative by flicking the tube.
10. Add 1ml cold fixative. Invert tube
11. Spin down in a microfuge for 2min. at 1500 RCF
12. Very gently remove most of the supernatant with a P1000, being careful not to
disturb the pellet. Gently resuspend in the residual fixative by flicking the tube.
13. Add 1ml cold fixative. Invert tube
14. Spin down in a microfuge for 2min. at 1500 RCF
15. Remove all but 50-100μl of fixative, and gently resuspend cells in the remainder.
16. Store at -20oC.