Mitotic chromosome preparations from embryo tail tips

Solutions needed

• RPMI medium 1640 (GIBCO 22400-097) 100ml (any culture medium will do)
• 0.56% KCl (0.075m) (0.56g in 100mL deionized H2O, freshly prepared)
• Fixative - acetic acid: methanol, 1:3.
• Colchicine (Sigma C3915-500mg)
• Stock solution: 0.5g colchicine / 100ml sterile distilled water.
• Working solution: 0.1ml of 0.5% colchicine in 9.9ml 9%NaCl. (50ug/ml)

Procedures

1. Collect embryos in PBS or RPMI1640; remove tail tip (e15.5), whole tail (10.5, 12.5).
2. Transfer tail tip to small petri dish or plate well ~0.5 ml of RPMI medium with 0.5ml of working solution colchicine added.
3. Mince tissue with fine scissors. (Omit this step with 10.5 and 12.5 embryos).
4. Transfer to 5-ml conical tube with syringe and 22g needle.
5. Further breakup tissue by pipetting in and out of 22g needle.
6. Allow to sit at room temperature until 20-30 minutes total time in medium with colchicine.
7. Spin, benchtop clinical centrifuge, 400g, 5minutes.
8. Pipet off medium; add 0.5ml of pre-warmed KCl; let sit at RT for 10-15minutes.
9. Spin, bench-top clinical centrifuge, 400g, 5minutes.
10. Pipet off KCl; and flick tube bottom gently; trying not to splash remaining liquid up side of tube; add 0.5ml fixative, 3:1 Methanol/glacial acetic acid. Allow to stand at RT about 20 mins. Can hold overnight in refrigerator at this point.
11. Spin as in step #7 and wash cells in 0.5ml fixative.
12. Spin once more and then re-suspend cells in about .25ml fixative and make air dry preps. The smaller the tail tip, the more one needs to be concerned about getting sufficient mitotic cells. Methods to maximize cells include (1) use small volumes, (2) avoid splashing sample up sides of tube, (3) use entire sample on slide.

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.