Mitotic chromosome preparations from bone marrow
Muriel T. Davisson
- 0.5% colchicine (Sigma C3915) in water (see recipe; store at 4°C)
- Stock Solution: Dissolve 0.5 g colchicine in 100 ml sterile distilled H20 = 0.5%
- 0.56% (0.075 M) KCl (0.56 g in 100 ml deionized H2O; freshly prepared)
- Fixative (3:1 methanol:glacial acetic acid; freshly prepared)
- 1 cc disposable syringes with 23 gauge needles
- 5 3/4" Pasteur pipets
- 15 ml conical centrifuge tubes (Corning #25310)
- IEC clinical bench top centrifuge with 221 rotor (or equivalent); max RCF=1625xg
EDTA buffer solution:
(for alternate method) with 0.025% colchicine
(For convenience and long-term storage, keep sterile.)
- 8.0 gm NaCl
- 0.2 gm KH2PO4
- 0.2 gm KCL
- 1.15 gm Na2HPO4
- 0.2 gm EDTA (diodium salt)
Dissolve in 1 liter of deionized water and autoclave. May freeze in 100mL aliquots. Add 500 ul 0.5% colchicine stock solution to 100 ml of EDTA buffer solution before using and prewarm to 37°C.
Bone marrow procedure
- Inject mouse with 0.1 cc of 0.5% colchicine (stock solution) intraperitoneally (see alternate method below). Wait 30 min.
- Wait time can be 20 min. in young mice and should never be longer than 1 hr. (see alternate method below).
- Sacrifice mouse and remove femur(s) and tibia(s). Early metaphases seem to be more prevalent in tibias.
- Cut off just enough of the bone heads to insert a 23-gauge needle into the marrow cavity.
- Flush out cells into a conical centrifuge tube using a 1 cc syringe filled with 0.56% KCl.
- Incubate the tubes at room temperature (or 37° C if room is cool) for 15 min.
- Centrifuge at 400 g for 5 min. in a clinical bench-top centrifuge.
- Remove supernatant and add 0.5 ml of fixative without disturbing the pellet. Remove fixative after 3-4 sec. and add 2 ml fresh fixative without disturbing the pellet.
- Allow tubes to sit at room temperature 30 min. The procedure can be interrupted at this point and resumed later. Always refrigerate cells if they are to be left standing in fixative longer than 30 min.
- After 30 min. centrifuge the cells at 400 g, remove the fixative, and resuspend the cells in fresh fixative.
- Repeat step 9 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.
- Remove bone marrow from femurs and tibias into approximately 3ml of 0.025% colchicine solution (prepared in EDTA buffer) using 1cc tuberculin syringe and 23g needle. Use 15ml conical centrifuge tube.
- Break up bone marrow using syringe and needle. Bring volume to 3ml and incubate (37°C) for 10 mins.
- Centrifuge at 400g for 5 minutes in a clinical bench-top centrifuge, remove supernatant and resuspend in 1.5mL 0.56% KCl.
Now follow above procedure at #5.