Protocol for Metaphase chromosome spreads
A. Solutions:
Hypotonic solution (0.8 % Sodium Citrate)
Add 8.0 g of Sodium Citrate to 1.0 L of deionized water
Incubate at 37˚C beforehand!
Carnoy’s Fixative (75 % Methanol, 25% Acetic Acid)***
Mix three parts of Methanol with one part of Glacial Acetic Acid
*** Must be made fresh just before using
1X Trypsin-EDTA
B. Protocol:
1. Add 100 l of 10 g/ml colcemid (final concentration 100 ng/ml) to a p100 dish
containing 10 ml of medium, incubate at 37˚C for 1.5 hours (the incubation can
range from 30 minutes to 1.5 hours depending on the cell type and mitotic index)
2. After the incubation, remove the medium from the p100 dish and transfer to a 15
ml conical tube
3. Rinse the p100 dish with 2 ml of 1X Trypsin-EDTA and transfer the 1X TrypsinEDTA to the 15 ml conical tube
4. Add 2 ml of 1X Trypsin-EDTA and incubate at 37oC for 5 minutes
5. Remove 2 ml from the 15 ml conical tube and add it to the p100 dish
6. Rinse the p100 dish and transfer the 1X Trypsin-EDTA to the 15 ml conical tube
7. Centrifuge at 1,000 rpm for 10 minutes at 4˚C
8. Aspirate most of the medium using a micropipet tip, leave 500 l behind
9. Flick the pellet carefully in the remaining medium to mix
10. Slowly (dropwise) add 7 ml of the hypotonic solution to each sample while gently
tapping the tube to mix (no vortexing at any time!)
11. Incubate at RT for 10 minutes
12. Centrifuge at 1,000 rpm for 10 minutes at 4˚C
13. Aspirate most of the supernatant using a micropipet tip, leave 500 l behind
14. Gently resuspend the pellet in the remaining supernatant
15. Slowly add 7 ml of Carnoy’s Fixative to each sample while mixing
16. Incubate at RT for 10 minutes
17. Centrifuge at 1,000 rpm for 10 minutes at 4˚C
18. Repeat steps 15-17 two additional times
19. Aspirate as much of the supernatant as possible and add 300-500 l of Carnoy’s
Fixative to resuspend the pellet
20. Make slides:
i. Turn a water bath to 37˚C
ii. Remove the top from an empty box of p1000 tips
iii. Place a wet paper towel in the box top and put it in the 37˚C water bath
iv. Place the top of a slide (Fisher SuperFrost*/Plus, catalog #12-550-15)
against one side of the box top (the slide should now be angled)
v. Use a glass pasteur pipet to resuspend the cells/fixative
vi. Place a stepping stool near the water bath
vii. Stand on the stepping stool and release 3 drops of the cells/fixative from
the pasteur pipet onto the slide (try to release the drops to different areas
of the slide)
viii. Prepare 3-4 slides for each sample and place in a chemical hood to dry
ix. Once slides dry they can be stained with Giemsa stain (Sigma Giemsa
Stain, catalog #GS-500, dilute in water 1:20) for 30 minutes
x. Rinse twice with ddH2O
xi. Air dry
xii. Add mounting medium to a cover slip and place this on top of the slide (be
careful not to get bubbles)
xiii. Once dry seal with nail polish
xiv. Take pictures