Preparation Of Normal Target Metaphase Chromosomes

Peripheral blood lymphocytes of normal female or male donors are cultured to prepare metaphase chromosomes. Cell division is induced using phytohemagglutinin. Colcemid treatment, which inhibits microtubule formation, causes mitotic arrest. The cellular membrane becomes fragile during hypotonic swelling of the cell. The metaphase chromosomes are fixed by dehydration and proteins are removed using a mixture of methanol and glacial acetic acid. Finally, the suspension is dropped onto clean glass slides and the spreading takes place when the methanol/acetic acid evaporates.

CAUTION: When working with human blood, cells, or infectious agents, biosafety practices should be followed; see unit 5.1.

Materials

RPMI 1640 medium (Life Technologies)

100x antibiotic-antimycotic: 10,000 U/ml penicillin G sodium, 10,000 |g/ml streptomycin sulfate, 25 |g/ml amphotericin B (Life Technologies) Fetal bovine serum (FBS): qualified, heat-inactivated, sterile-filtered (Life

Technologies) Phytohemagglutinin (PHA; Murex Diagnostics Ltd.) Normal whole blood (heparin anticoagulated) 10 |g/ml KaryoMAX colcemid solution (Life Technologies) 0.4% (w/v) KCl, 37°C

3:1 (v/v) methanol/glacial acetic acid fixative, freshly prepared 1:1 (v/v) ethanol/ether 70%, 90%, and 100% ethanol

75-cm2 tissue culture flasks 50-ml and 15-ml centrifuge tubes 37°C and 60°C water bath Microscope slides

NOTE: All incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

Process blood and coat culture flask

1. Prepare a 75-cm2 tissue culture flask containing 40 ml RPMI 1640 medium supplemented with 0.4 ml antibiotics and 8 ml FBS. Add 400 |l PHA.

2. Collect 10 ml whole blood in a tube with heparin as anticoagulant. Centrifuge 10 min at 300 x g, room temperature, or allow to settle 2 to 3 hr at room temperature. Discard serum and save lymphocyte layer (buffy coat) just above the erythrocytes.

3. Add 2 ml lymphocyte layer to the 75-cm2 flask from step 1.

Final volume in the flask is 50 ml.

Culture and process lymphocytes

4. Culture 72 hr in a 37°C, 5% CO2 incubator. Shake flasks once a day.

5. Add 500 |l of 10 |g/ml KaryoMAX colcemid solution and mix well but gently by shaking or pipetting.

6. Immediately transfer into two 50-ml centrifuge tubes and incubate 20 min at 37°C.

7. Centrifuge 12 min at 300 x g, room temperature. Remove supernatant, leaving at least 5 ml in tube, and resuspend pellet by vortexing or pipetting until it is completely dissolved.

8. Add 5 ml prewarmed 0.4% KCl to each tube and mix by shaking tube, vortexing, or pipetting up and down.

9. Add more 0.4% KCl to a total of 40 ml. Mix well. Incubate 10 min in a 37°C water bath.

10. Repeat steps 7 and 8.

Fix cells

11. Add 5 ml freshly prepared 3:1 methanol/glacial acetic acid fixative per tube and mix by shaking tube, vortexing, or pipetting up and down.

12. Add more 3:1 methanol/glacial acetic acid fixative to a total of 25 ml per tube.

13. Repeat steps 7, 11, and 12 two more times.

14. Transfer cell suspension to 15-ml centrifuge tubes.

15. Repeat steps 7, 11, and 12 using 10 ml of 3:1 methanol/glacial acetic acid fixative four more times, always leaving at least 2 ml in tube.

16. Resuspend pellet in 4 to 6 ml of 3:1 methanol/glacial acetic acid fixative.

Prepare slides and observe chromosomes

17. To prepare slides, dip in 1:1 ethanol/ether or 3:1 methanol/glacial acetic acid fixative and wipe with paper towels. Drop ~100 |l cell suspension onto a clean slide.

18. Check the number of metaphases, the spreading of the chromosomes, and the amount of cytoplasm left around the metaphases. Adjust dropping conditions; change temperature, humidity, and dilution.

Frequently, good results are obtained by placing the slides on a rack in a 60°C water bath, dropping ~100 ^l per slide. Alternatively, drop suspension onto slides and cover with foil

Molecular Cytogenetics

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