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SUSANN NEUBAUER
hand by quantifying the mis- and unrepaired break-events visible as chromosomal aberrations, and on the other hand by the frequency of complex
aberrations (Neubauer et al. 1997).
Outline
1. Heparinized peripheral blood of each patient is divided into (at least)
three portions. One sample remains unirradiated and serves as a control for spontaneous frequency of aberrations, the other two (or more)
samples are irradiated in vitro with 0.7 and 2.0 Gy of X-rays.
2. After exposure, classical lymphocyte cultures are set up and grown at
37°C for 48 h.
3. 4 h before standard metaphase spreads are prepared, colcemid is added
to induce a mitotic block.
4. Chromosome suspension is dropped onto slides and air dried slides
are kept in 70% ethanol at -20°C before being subjected to the painting
procedure.
5. Before performing the three-color FISH assay, a slide pretreatment
is carried out to remove cell debris and improve the quality of the
metaphase spreads.
6. Denaturation of the slides is performed for 1 min at n°e. The biotinylated and/or digoxigenated whole chromosome probes are denatured for 5 min and hybridized on the slides for two nights at 37°C
in a humid chamber. After a postwashing series, detection of biotin
with avidin-fluorescein-isothiocyanate (=FITC) and digoxigenin with
anti-digoxigenin-rhodamine and a final counterstaining with DAPI
finishes the painting procedure. Thus, chromosome I is painted in
red, chromosome 2 in green and the mixture of both labels leads to
a yellow color of chromosome 4, unpainted chromosomes appear
in a blue color.
7. Evaluation is done on a fluorescence microscope with suitable fIlter
combinations (FITC/Texas Red/DAPI). Chromosomal rearrangements
(characterized mainly by a color transfer to other chromosomes -
Fig. I) are classified according to standard cytogenetic criteria from
1000 (unirradiated), 400 (0.7 Gy) and 200 (2.0 Gy) metaphases per
case. Aberration frequencies are expressed as a number of breakpoints
per mitosis (B/M). The frequencies of break events were estimated
SUSANN NEUBAUER
hand by quantifying the mis- and unrepaired break-events visible as chromosomal aberrations, and on the other hand by the frequency of complex
aberrations (Neubauer et al. 1997).
Outline
1. Heparinized peripheral blood of each patient is divided into (at least)
three portions. One sample remains unirradiated and serves as a control for spontaneous frequency of aberrations, the other two (or more)
samples are irradiated in vitro with 0.7 and 2.0 Gy of X-rays.
2. After exposure, classical lymphocyte cultures are set up and grown at
37°C for 48 h.
3. 4 h before standard metaphase spreads are prepared, colcemid is added
to induce a mitotic block.
4. Chromosome suspension is dropped onto slides and air dried slides
are kept in 70% ethanol at -20°C before being subjected to the painting
procedure.
5. Before performing the three-color FISH assay, a slide pretreatment
is carried out to remove cell debris and improve the quality of the
metaphase spreads.
6. Denaturation of the slides is performed for 1 min at n°e. The biotinylated and/or digoxigenated whole chromosome probes are denatured for 5 min and hybridized on the slides for two nights at 37°C
in a humid chamber. After a postwashing series, detection of biotin
with avidin-fluorescein-isothiocyanate (=FITC) and digoxigenin with
anti-digoxigenin-rhodamine and a final counterstaining with DAPI
finishes the painting procedure. Thus, chromosome I is painted in
red, chromosome 2 in green and the mixture of both labels leads to
a yellow color of chromosome 4, unpainted chromosomes appear
in a blue color.
7. Evaluation is done on a fluorescence microscope with suitable fIlter
combinations (FITC/Texas Red/DAPI). Chromosomal rearrangements
(characterized mainly by a color transfer to other chromosomes -
Fig. I) are classified according to standard cytogenetic criteria from
1000 (unirradiated), 400 (0.7 Gy) and 200 (2.0 Gy) metaphases per
case. Aberration frequencies are expressed as a number of breakpoints
per mitosis (B/M). The frequencies of break events were estimated