ES cells Core facility
Samuel Lunenfeld Research Institute Mount Sinai Hospital
600 University Avenue Toronto Ontario M5G 1X5
416-586-4519
Counting ES cells chromosomes
The normal mitotic karyotype of mouse cells consists of 40 acrocentric chromosomes.
After the conventional staining described below chromosomes show few distinguishing
morphological features not sufficient for complete analysis that requires chromosome
banding. However, the procedure is suitable for chromosomes counting as an indication
of the potential of ES clone for generating germ line chimeras.
Equipment and solutions
Pre-cleaned microscope slides (e.g. Fisherbrand Superfrost cat. # 12-550-12 or #12-549)
Microscope cover glass 22x50 (e.g. ESBE Scientific cat. # ESO2250B)
Coplin staining jars
Microscope with 100x oil immersion and 40x phase-contrast objectives
Hypotonic solution (0.075M Potassium Chloride (0.559 g KCl in 100 ml water)
– pre-warmed @ 370C before use
Colcemid 10ug/ml (Gibco # 15212-012)
Gurr’s buffer tablets (pH 6.8) (Gibco # 10582-013 or BDH 33-193)
Giemsa stain (Gibco cat. # 10092-013)
Fixative (made up fresh each time and disposed properly into a solvent waste):
3 parts methanol to 1 part glacial acetic acid – kept @40C before use.
Procedure
An actively growing culture is required, i.e. 24 or 48 hours after usual 1:5 passage on
gelatinized 60mm plate.
1. Culture cells for 1 to 1.5 hours in the presence of Colcemid (25 ul 10 ug/ml stock
solution per 5 ml of media -> 0.05 ug/ml final concentration). To accumulate very
large number of mitotic figures, it is possible to incubate the culture in Colcemid for
3-4 hours. Chromosomes will be short and suitable only for counting.
2. Remove the media, wash with PBS, trypsinize for 3-5 minutes the usual way.
3. Harvest cells into a 15 ml conical centrifuge tube, centrifuge for 5 minutes, aspirate
the supernatant and flick the pellet; Resuspend the cells in 1.5 ml of fresh media.
4. Add 10 ml of warmed KCl solution slowly; drop by drop, along the wall of the tube,
flicking the tube gently at the same time. Invert several times, and incubate at 370C
for 15-20 minutes – the time in KCl is crucial. If it is too short, the chromosomes
will be too tightly packed with cytoplasm present around metaphases; too long,
they will not be contained in their appropriate group.
5. Add 2-3 drops of cold fixative, invert the tube, centrifuge on 4 for 5 minutes;
6. Aspirate supernatant, leaving ~1 ml behind, flick the pellet.
7. Fume hood from this step: Using vortex add up to 10 ml of cold fresh fix drop by
drop using vortex to disperse cells thoroughly in the fix; Centrifuge for 5 minutes;
Aspirate supernatant, leaving ~ 1 ml behind, flick the pellet.
8. Repeat step 7 at least one more time or better 2-3 times for total of 3-4 fixations.
ES cells Core facility
Samuel Lunenfeld Research Institute Mount Sinai Hospital
600 University Avenue Toronto Ontario M5G 1X5
416-586-4519
9. Slides are best made within 3 hours of the final fixation, but can be made later if cells
are kept in fixative at 40C or –200C and resuspended in fresh fixative before making
spreads.
10. Resuspend the cells in a small volume of fixative (~0.5 ml) that can be diluted later if
the suspension is too dense. 4-5 slides should be made from each sample.
11. Prepare few slides in water in Coplin jar. Resuspend cells using yellow tip. Make
spreads by slowly dispersing 80 ul of cell suspension on a wet slide in a moist
condition, put few drops of fixative on top of the slide. Air dry the slides in moist
condition. Home made water bath (bucket with hot water and empty tips rack placed
inside and kept in the hood) helps to maintain the humidity during slide preparation if
it is too dry in the lab. To assist drying, slides can be heated briefly at desk lamp bulb
and by blowing at them from the mouth at the same time.
12. Scan the first slide with 40x objective, dilute the suspension if necessary.
13. Stain slides for 10 minutes in Coplin jar with freshly made Giemsa stain (2.5 ml of
Giemsa stain in 47.5 ml of Gurr’s pH6.8 buffer). Rinse with water in Coplin jar until
clear. Air dry.
14. Coverslip using available mounting media (e.g. Entellan BDH #65037-71).
15. Count with 100x oil immersion objective. Look for spreads in widely separated fields.
Ignore spreads with fewer than 39 chromosomes. 20 + good spreads should give
reasonable sampling. ES lines with minimum 70 % of spreads containing 40
chromosomes are acceptable.