5.2.1 Chromosomal analysis
220.127.116.11 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.
- Add to cell culture medium 0.125 µg/ml colchicin and incubate for 5-6 hours at 28C.
- Treat monolayer of culture cells with one drop of Ca/Mg-free PBS containing 0.1% trypsin and 0.01% EDTA for 1 min.
- Add 200 µl of the culture medium and gently pipette up and down to form cell suspension.
- Transfer the cell suspension to an 1.5ml tube and centrifuge it at 1000 rpm for 6 minutes.
- Remove the supernatant and resuspend the pellet in 250 µl of hypotonic KCL solution (0.075 M) and incubate at 26C for 6 minutes.
- 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.
- 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.
- Repeat step7 once or twice more after 30 minutes and keep the cell suspension at -20 C.
- 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.
- Place 3 drops of the suspension onto a clean slideglass that has been kept in cold (4 C) distilled water.
- Dry the slide at room temperature.
- Stain the slide with 3% Giemsa solution for 10 minutes.
- Rinse in distilled water and dry in air.
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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