Mitotic chromosome preparations from 5 days old newborns
Muriel T. Davisson
Culture medium - any kind.
0.56% KCl (0.075m) (0.56g in 100mL deionized H20, freshly prepared)
Fixative - acetic acid: methanol, 1:3.
Fixative (optional) - acetic acid: methanol, 1:6.
EDTA Buffer Solution with 0.025% colchicine (For convenience and long term storage keep sterile)
8.0 gm NaCl
0.2 gm EDTA (diodium salt)
1 liter H20
0.2 gm KCl
0.2 gm KH2PO4
1.15 gm Na2HPO4
Dissolve in 1 liter of deionized H2O and autoclave. May freeze in 100ml aliquot. Add 500 ul of 0.5% colchicine stock solution to 100 ml of the EDTA buffer solution just before use and prewarm to 37°C.
1 To remove livers place forceps on both sides of liver and pinch so liver will pop up through skin. Clean away extraneous tissue and place in culture medium (prewarmed to 37°C). Wash once to remove blood and debris. Place cleaned liver into small petri dish with 1 pipetful of medium.
2 Mince tissue with Dewecker scissors. Pipet several times against bottom of dish with broken tip Pasteur pipet to break up tissue more. Try not to create any bubbles as cells may get trapped in them. Tilt petri dish to allow chunks of tissue to settle. Transfer supernatant with suspended cells (leaving tissue pieces) to conical centrifuge tubes.
3 Centrifuge 5 minutes at 400 x g on clinical benchtop centrifuge. Add 1 pipetful of colchicine solution (in EDTA) Incubate @ 37°C for 10 mins. Centrifuge at 400 x g for 10 mins. Colchicine disrupts spindle formation so the chromosomes will spread better.
4 Remove supernatant and discard. Resuspend pellet by flicking bottom of tube with finger. Add 1 pipetful of hypotonic (0.56%) KCl (prewarmed to 37°C). Gently resuspend pellet in KCl solution and let stand at room temperature 12-15 minutes (longer time in KCl ruptures the cells so the chromosomes are scattered).
5 Centrifuge as before and remove supernatant. Add 1/2 pipetful of fixative (1:3 glacial acetic acid: methonal) without disturbing the pellet and remove. Do this 2 times to remove all the KCl. Add 1/2 pipetful of fresh fixative and let stand 5-10 minutes or until pellet is completely white.
6 Remove supernatant. Resuspend pellet by flicking bottom of tube with finger. Add 1/2 pipetful of fresh fixative and pipet gently. Centrifuge as before. Wash cells 2 times more.
7 After 2nd washing, remove supernatant and add fixative to dilute the cell suspension for making slides (may use fresh 1:6 fixative for better chromosome spreading). Let larger clumps settle before making slides. Do not remix cell suspension.
8 Chromosome Slide Preparation
a. Immerse pre-cleaned slides in fixative at least 15 min. prior to use. Wipe slides dry with a Kimwipe or other lint-free tissue.
b. 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.
c. 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.
d. Repeat steps b and c until sufficient sample is on the slide. Monitor cell concentration, cell spreading, and contrast by phase microscopy.