Mitotic chromosome preps from liver

Muriel T. Davisson

Materials needed

             Culture medium, any kind

             0.5% colchicine (Sigma C3915) in water (see recipe; store at 4°C) or

             0.56% (0.075 M) KCl (0.56 g in 100 ml deionized H2O; prepare fresh)

             Fixative (3:1 methanol:glacial acetic acid; prepare fresh)

             Fixative (6:1 methanol:glacial acetic acid; prepare fresh; optional for making slides)

             Small Petri dish (60 mm)

             Small glass dish (~35 mm)

             Dewecker iris scissors for mincing tissue

             9" Pasteur pipets

             15 ml conical centrifuge tubes (Corning #25310)

             IEC clinical bench top centrifuge with 221 rotor (or equivalent); max RCF=1625xg

Reagents and solutions

Colchicine stock solution: Dissolve 0.5 g colchicine in 100 ml sterile deionized H2O = 0.5%.

EDTA buffer solution with 0.025% colchicine (for in vitro method)

             8.0 gm NaCl

             0.2 gm EDTA (diodium salt)

             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.

Culture procedure

1    a. Inject pregnant female 10-15 minutes ahead with 0.1cc of 0.5% colchicine (stock solution) intraperitoneally. Remove livers from 14-day embryos.  For younger embryos any internal tissue will do. 15 day and older embryos have fewer metaphases. Colchicine disrupts spindle formation so the chromosomes will spread better.

     b. In vitro method: If you do not want to inject the pregnant female, collect cells as described in steps 2-3 below; remove supernatant and incubate the re-suspended cells in 1 ml of EDTA buffer with 0.025% colchicine for 10 min at 37°C. Proceed to step 4.

2    To remove livers, place forceps on both sides of the liver and pinch so that the liver will pop up through the skin. Clean away any extraneous tissue and place the liver in a Petri dish with culture medium (pre-warmed to 37°C). Wash once to remove any blood and debris. Place cleaned liver into a small (~35 mm diameter) glass dish with 1 pipetful of medium.

3    Mince tissue with Dewecker iris scissors. Pipet several times against the bottom of the dish with a broken tip Pasteur pipet to break up tissue more. Try not to create any bubbles as the cells may get trapped in them. Tilt the dish to allow the chunks of tissue to settle. Transfer supernatant with suspended cells (leaving tissue pieces) to a conical centrifuge tube.

4    Centrifuge 5 minutes at 400 g in a clinical bench top centrifuge.

5    Remove supernatant and discard. Re-suspend pellet by flicking bottom of tube with finger. Add 1 pipetful of hypotonic (0.56%) KCl (pre-warmed to 37°C). Gently re-suspend the pellet in the KCl solution and let stand at room temperature 12-15 minutes.  Longer time in KCl ruptures the cells so the chromosomes are scattered.

6    Centrifuge as before and remove supernatant. Add 1/2 pipetful of fixative without disturbing the pellet and remove. Do this twice to remove all the KCl. Add 1/2 pipetful of fresh fixative and let stand 5-10 minutes or until the pellet is completely white.

7    Remove supernatant. Re-suspend pellet by flicking bottom of tube with finger. Add 1/2 pipetful of fresh fixative and pipet gently. Centrifuge as before.

8    Wash cells twice, as above.

9    Remove supernatant and add fixative to dilute the cell suspension for making slides (solution in tube should look slightly opaque). Let larger clumps settle before making slides. Do not remix cell suspension. Fresh 1:6 fixative may be used for better chromosome spreading.

10  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.   

References

Evans, E.P. 1987. Karyotyping and sexing of gametes, embryos and fetuses and in situ hybridization to chromosomes. In Mammalian Development: A Practical Approach (M. Monk, ed.) pp. 93-114. Oxford University Press, Oxford.

Akeson, E.C., Davisson M.T.  2000.  Mitotic chromosome preparations from mouse cells for karyotyping unit 4.10.  In Current Protocols in Human Genetics (Suppl 25) 4.10.1-4.10.19.