23 Detection of Radiosensitivity by Means of Three-Color FISH
293
as the minimum number regarded to be necessary for the formation of
the observed aberrations.
Procedure
Irradiation procedures and lymphocyte cultures
1. Heparinized freshly drawn peripheral blood is irradiated with different
doses (0.7 and 2.0 Gy) of 6 MV X-rays from a linear accelerator (Mevatron) at a dose rate of 2.2 Gy/min.
2. 1 ml of whole peripheral blood is stimulated in 9 ml RPMI 1640 culture
medium supplemented with 15% foetal calf serum, 2.5% phytohemagglutinin standard solution, 1% penicillin-streptomycin (10,000 IU/ml).
3. 180 III Co1cemid (0.09 1l1lml) is added to each blood culture 4 h before
harvesting to block mitoses.
4. Chromosome preparations are carried out according to standard procedures (see. e.g. Chapter 3 by Liehr and Claussen).
5. Air dried slides are kept in 70% ethanol at -20°C before being subjected
to the three-color FISH painting.
Pretreatment of metaphase spreads on slides
Pretreatment is useful when the quality of chromosome preparation is
suboptimal or the metaphase spreads are covered with cytoplasm. Pretreatment with RNase and pepsin can remove cell debris and significantly
improve the quality of the metaphase spread. All washing procedures
mentioned below are performed in a 100 ml Coplin jar with a maximum
of 7 slides and shaken gently.
1. Take the slides from the 70% ethanol storage solution and air dry.
2. Pipette 100 III of RNase A solution (50 mglml in 2xSSC) onto each slide
and cover with a coverslip for 15-30 min at 37°C in a humid chamber.
The incubation time is dependent on the cell density and cytoplasm
present on the slide. The slides can be looked at by light microscopy
and treatment can be repeated when the outcome is not satisfactory.
293
as the minimum number regarded to be necessary for the formation of
the observed aberrations.
Procedure
Irradiation procedures and lymphocyte cultures
1. Heparinized freshly drawn peripheral blood is irradiated with different
doses (0.7 and 2.0 Gy) of 6 MV X-rays from a linear accelerator (Mevatron) at a dose rate of 2.2 Gy/min.
2. 1 ml of whole peripheral blood is stimulated in 9 ml RPMI 1640 culture
medium supplemented with 15% foetal calf serum, 2.5% phytohemagglutinin standard solution, 1% penicillin-streptomycin (10,000 IU/ml).
3. 180 III Co1cemid (0.09 1l1lml) is added to each blood culture 4 h before
harvesting to block mitoses.
4. Chromosome preparations are carried out according to standard procedures (see. e.g. Chapter 3 by Liehr and Claussen).
5. Air dried slides are kept in 70% ethanol at -20°C before being subjected
to the three-color FISH painting.
Pretreatment of metaphase spreads on slides
Pretreatment is useful when the quality of chromosome preparation is
suboptimal or the metaphase spreads are covered with cytoplasm. Pretreatment with RNase and pepsin can remove cell debris and significantly
improve the quality of the metaphase spread. All washing procedures
mentioned below are performed in a 100 ml Coplin jar with a maximum
of 7 slides and shaken gently.
1. Take the slides from the 70% ethanol storage solution and air dry.
2. Pipette 100 III of RNase A solution (50 mglml in 2xSSC) onto each slide
and cover with a coverslip for 15-30 min at 37°C in a humid chamber.
The incubation time is dependent on the cell density and cytoplasm
present on the slide. The slides can be looked at by light microscopy
and treatment can be repeated when the outcome is not satisfactory.