5.2.1 chromosomal analysis of primary cultured cells from the caudal fin

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5.2.1 Chromosomal analysis

Elena Kaftanovskaia
Nagoya University

5.2.1.0 Preparation of culture cells

A commercially available orange-red variety of the medaka is used. The small tissue fragments of the caudal fin of adult fish are used for primary cultures. Actively proliferated cells cultured for 4 to 6 days are used for chromosome preparations.

5.2.1.1 Procedure

1. Add to cell culture medium 0.125 µg/ml colchicin and incubate for 5-6 hours at 28C.
2. Treat monolayer of culture cells with one drop of Ca/Mg-free PBS containing 0.1% trypsin and 0.01% EDTA for 1 min.
3. Add 200 µl of the culture medium and gently pipette up and down to form cell suspension.
4. Transfer the cell suspension to an 1.5ml tube and centrifuge it at 1000 rpm for 6 minutes.
5. Remove the supernatant and resuspend the pellet in 250 µl of hypotonic KCL solution (0.075 M) and incubate at 26C for 6 minutes.
6. Add one drop of freshly prepared ice-cold fixative (3 parts of methanol : 1 part of acetic acid) to the cell suspension and gently mix.
7. Immediately centrifuge it at 1000 rpm for 6-7 minutes, remove the supernatant, and resuspend the pellet in 200 µl of the fresh cold fixative.
8. Repeat step7 once or twice more after 30 minutes and keep the cell suspension at -20 C.
9. Centrifuge the fixed cell suspension at 1000 rpm for 6 minutes, remove the supernatant, and resuspend the pellet in 50 µl of the fresh cold fixative.
10. Place 3 drops of the suspension onto a clean slideglass that has been kept in cold (4 C) distilled water.
11. Dry the slide at room temperature.
12. Stain the slide with 3% Giemsa solution for 10 minutes.
13. Rinse in distilled water and dry in air.

5.2.1.2 Figure

The attached photograph is a diploid (2n=48) chromosome spread of culture cell derived from caudal fin of orange red adult fish.

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