Mitotic Chromosome from Cell Cultures

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Mitotic Chromosome from Cell Cultures

Solutions Needed:

Colchicine (Sigma # C3915) 0.5g colchicine/100ml sterile distilled water.
Hypotonic saline (KCI) = 0.56% potassium chloride in distilled water.
Fixative = methanol: glacial acetic acid, 3:1.
Colchicine Treatment
Monolayer cultures:

Add 0.1 ml of colchicine (50 g/ml) per ml of cell culture medium to the proliferating cell culture.
Incubate at 37°C for 30 - 60 min. (depending on how rapidly the cells are dividing).
Detach the cells from the flask by scraping with a rubber policeman. Do not use trypsin to dissociate the cells because it can interfere with the chromosomal banding.
Cell Suspensions:

Add 0.2 ml colchicine (50 g/ml) per 2 ml of cell suspension (final cell suspension should be approximately 2.4 x 106 cells).
Incubate at 37°C for 30 - 60 min.
Cell Harvest:

Transfer cell suspension to a conical centrifuge tube and centrifuge for 5 minutes in a clinical bench-top centrifuge at 400 x g.
Remove supernatant and gently add 2-3 ml of warm (37°C) hypotonic KCI (0.56%). Gently resuspend cells by pipetting. Incubate at 37°C for 15 - 20 minutes.
Centrifuge 5 min. at 400 x g
Remove supernatant without disturbing pellet. Gently add 3-4 mls fixative down side of tube. Then pipet gently but rapidly to prevent cell clumping. Fixative = methanol: glacial acetic acid, 3:1.
Stopper tube and allow to sit for at least 30 min. Refrigerate if tubes are to be held more than 30 min. The procedure can be interrupted at this point and resumed later.
After 30 min. centrifuge cells at 400 x g, pipet off fixative and resuspend in fresh fixative. Repeat this step twice more. The last addition of fixative should be just enough to make a thin cell suspension (solution in tube will look slightly opaque).
Chromosome Slide Preparation

Immerse pre-cleaned slides in fixative at least 15 min. prior to use. Wipe slides dry with a Kimwipe or other lint-free tissue.
Drop small drops of cell suspension onto slide surface with a Pasteur pipet and allow it to spread. If too much suspension is used it will bubble at the edges.
As soon as the drop begins to contract and Newton's rings are visible (rainbow colors around the edge of the drop), blow on the slide surface to accelerate drying. Rapid drying is critical to obtaining well spread metaphases. Slides may also be dried by using tubing connected to an air supply or by spraying lightly with Dust-off Plus. The "bomb" method of dropping one drop of fixative onto the cell suspension once it has started to dry may be used to increase the spreading of the chromosomes. Use a very small drop of fixative and keep the slide in a flat position while adding the fixative and until the slide is completely dry. Do not blow on the slide.
Repeat steps b and c until sufficient sample is on the slide. Monitor cell concentration, cell spreading, and contrast by phase microscopy.