An actively growing culture of cells is required, i e 2 - 3 d ES cell culture. The total number of cells needs to be between 106 - 107 cells.
N B Read notes at end of method.
1) Harvest cells in the usual manner, into a conical bottom centrifuge tube.
2) Centrifuge to remove medium.
3) Resuspend cell button by flicking the bottom of the tube.
4) Add 8 ml of warmed KC1 solution to each tube and mix gently with a pipette.
5) Return to 37oC water bath for 10 - 14 mins, (this needs to be determined for each cell type)
6) Add 2 ml of fixative and mix by inversion.
7) Centrifuge cultures at 1500 rpm for 5 mins.
8) Aspirate most of the supernatant.
9) Using a pasteur pipette gradually resuspend the cells in ~2 mls of fixative. This needs to be done very gently. Then add fixative to 8 mls, and mix gently by inverting the tube.
10) Centrifuge cells again.
11) Repeat 8, 9, 10 3 - 4 times.
12) Gently resuspend cells at appropriate concentration.
13) Make spreads by "huffing" on slides, holding at 45oC and dropping one drop of cells onto the top of the slide.
Stain slides with Leishmanns.
STAINING
1) Stain slides with 1 ml Leishmann's stain and 5 ml pH 6.8 buffer for 7 - 9 mins (enough for 2 slides).
2) Rinse briefly in running water and air dry.
3) Clear in X2 changes of xylene.
4) Coverslip using Depex mountant.
SOLUTIONS.
HYPOTONIC SOLUTION (0.075M POTASSIUM CHLORIDE)
Add 0.56 gm KC1 to 100 mls H20.
LEISHMANN'S STAIN
BDH PROD 35022
0.2% w/v solution in methanol.
GURRS BUFFER. TABLETS pH 6.8
BDH PROD 33193
FIXATIVE
3 parts methanol to 1 part glacial acetic acid.
NOTES
The time in KC1 is crucial. If the time is too short, the chromosomes will be too tightly packed. Too long, they will not be contained in their appropriate group.
When staining spreads, the Leishmanns must be made up fresh for 2 - 4 slides at a time, as it precipitates out.
Slides need to be soaked in absolute ethanol overnight and wiped with a lint-free tissue. If it is too warm to "huff" on the slides, they can be placed in the fridge/freezer.
Fixative must be made up fresh each time. Dispose of fixative waste properly, i e into solvent waste.
Avoid breathing vapour.
