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THOMAS LIEHR
Outline
1. 1 ml of uncultured EDTA-, sodium acetate-treated or heparinized
blood or bone marrow aspirate is washed in cell culture medium, subjected to a hypotonic treatment and fIxed and washed in methanol/glacial acetic acid (3:1). The resulting suspension is dropped on slides,
which are incubated over night at room temperature.
2. For a FISH approach, these slides are treated with RNase and pepsin
prior to denaturation.
3. Denaturation of the slides is performed for about S min at 7S°C. Biotinylated and/or digoxigenated probes are denatured for S min and
hybridized on the slides for 3 nights at 37°C in a humid chamber. After
a postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to a green signal and of the
digoxigenated probe with anti-digoxigenin-rhodamine leading to red
signals. After counterstaining of the nuclei with DAPI and addition of
antifade solution, slides can be evaluated on a fluorescence microscope.
Procedure
Preparation of uncultured blood or bone marrow
1. Add 1 ml of blood or bone marrow (EDTA-, sodium acetate-treated or
heparinized) to 9 ml of cell culture medium (e.g. RPMI 1640 medium)
and mix the solution carefully.
2. Centrifuge the solution in a IS-ml tube at room temperature (= RT) for
8 min at 1000 rpm and discard the supernatant by sucking it off carefully with a glass pipette (1 ml of supernatant is left in the tube to avoid
loss of material).
3. For hypotonic treatment the pellet is resuspended in 10 ml 0.4% KCI
(37°C) and incubated at 37°C for 20 min.
4. Slowly add 0.6 ml of fIxative (= methanol/glacial acetic acid 3:1) (4°C)
and mix the solution carefully.
5. Repeat step 2.
6. Resuspend the peBet in 10 ml of fIxative (4°C) and incubate at 4°C for
20 min.