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

This is the protocol I use for wildtype MEFs. It may require some
adjustments for other genotypes or cell types.
1. Plate cells and grow to about 50-60% confluence.
2. Remove medium and add fresh medium containing 10μM BrdU.
3. Grow 18 hours. Its probably best to choose a few timepoints that bracket
18 hours if possible.
4. 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.
5. Trypsinize cells as normal and wash 1X 10ml PBS. At this point it is no
longer necessary to be sterile.
6. Remove as much PBS as possible and gently resuspend the cells in the
residual.
7. 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.
8. Incubate at 37oC (in a water bath) for EXACTLY 6 minutes.
9. Centrifuge at 900rpm for 5 minutes.
10. Remove as much KCl as possible and gently resuspend the cells in the
residual.
11. 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.
12. Centrifuge at 900rpm for 5 minutes and remove fixative
13. Slowly add 2 ml fixative dropwise.
14. 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.
15. Drop a few drops from about 18 inches high onto angled, humidified
microscope slide.
16. IMMEDIATELY blow on the slide very gently.
17. Air dry at least 10 minutes. Slides are now stable for a very long time.
18. Place slides into a staining jar containing about 70 ml 1XPBS+250 μl
Acridine Orange (20mg/ml in H20). Incubate 5 minutes in the dark.
19. Wash with several changes of 1XPBS, incubating about 5 minutes per
wash.
20. Allow excess liquid to run off and tap slide a few times on a paper towel
to remove residual liquid.
21. Place a few drops of Vectashield Iwithout DAPI) onto slide. Place
coverslip on slide and press gently.
22. Seal coverslip at short edges with nail polish and remove excess
Vectashield by aspirating.
Store slides at 4oC. They should be stable for a couple of weeks if stored in
the dark.
Solutions
0.075M KCl. Not necessary to filter or autoclave.
Freshly prepared Methanol/Acetic acid fixative. Add 1 part glacial
Acetic acid to 3 parts Methanol and chill at -20oC.
10mg/ml BrdU in PBS. Dissolve BrdU in PBS then filter using a 0.2μm
filter. Store in dark at -20oC
20mg/ml Acridine Orange in H2O. Not necessary to filter. Store in dark
at -20oC