Preparation of Lymphocyte Chromosomes
Culture
1. Prepare the following culture medium:
100 ml McCoy’s 5A medium with L-glutamin
25 ml foetal bovine serum
3.4 ml phytohemagglutinin
1.0 ml penicillin/streptomycin 10 000 U/ml
Keep the 10 ml medium in sterilised glass bottles at –20 ºC. Thaw bottles just before using.
2. Add 10 drops of blood (fresh or from Na-heparin solution) and mix.
3. Close bottle and incubate at 37 ºC for 72 h.
Harvest
1. Add 0.1 ml 10 g/ml colcemid 30 min before harvesting.
2. Centrifuge at 1000 rpm for 10 min.
3. Remove the supernatant and resuspend the cells in 0.075 M KCl. Add 6-7 ml KCl
while stirring the sample vigorously.
4. Leave at room temperature for 15 min.
5. Centrifuge at 1000 rpm for 10 min.
6. Remove the supernatant and resuspend the cells in fresh fixative (4:1
methanol:acetic acid). Add 5 ml of fixative while stirring the sample vigorously.
7. Centrifuge at 1000 rpm for 10 min.
8. Remove the supernatant and resuspend the cells in fresh fixative (3:1
methanol:acetic acid).
9. Centrifuge and repeat the fixation at least twice. Then keep in fridge, in freezer, or
make preparations directly as follows:
Slide preparation
1. Centrifuge the preparations at 1000 rpm for 10 min. Put the microscope slides in a
coplin jar with cool water.
2. Remove the supernatant and resuspend the cells in approximately 0.1 ml fresh
fixative (3:1 methanol:acetic acid).
3. Put 1-2 drops of the suspension on a slide and let it dry.
4. Check the quality through a phase contrast microscope. If the chromosome
spreading is insufficient, the fixative should be changed again or the acetic acid
concentration should be increased.
The preparations can be preserved at –20 ºC for years.