80
THOMAS LIEHR and UWE CLAUSSEN
Comments
1. In this chapter only the preparation of peripheral blood metaphase
spreads is described. For more specific preparation protocols (e.g.
for bone marrow or solid tumors) see more specialized handbooks
(e.g. Wegner 1999).
2. It is a well-known fact that EDTA- or sodiumacetate-treated blood
samples can not be successfully cultured in short-term culture.
3. Sterile cell culture conditions have to be kept, so as not to contaminate
the short-term culture.
4. BrdU is a thymidine analogue which is readily incorporated into chromosomes. Cultures containing BrdU should be protected from light,
otherwise chromosome breaks will occur. Moreover, the yield of metaphase may be reduced compared to cultures without BrdU, as this
chemical is cytotoxic.
5. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations can be too stringent, resulting in clean slides without any remaining metaphase spreads or nuclei.
6. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. If longer storage is necessary, slides are stable at -20°C
for several months.
7. Denaturation times of 2-3 min only will still allow the metaphase chromosomes to be evaluated. If only interphase nuclei are to be analyzed,
longer denaturation times of up to 6 min can lead to stronger and better estimable results.
8. In the current protocol the most common indirect probe-ligands, biotin and digoxigenine and one example of a detection system for them
are described. However, there are many other possibilities for directly
labeled ligands (like SpectrumGreen, SpectrumOrange, SpectrumAqua, etc.) or detection possibilities for the indirect ones (like CyS,
Cy5.5, TexasRed, etc.). The corresponding protocols for detection
are supplied by the companies and distributors and the chosen detection system mainly depends on the available fIlter sets of the own fluorescence microscope.
9. Incubation can be stopped - if necessary - after 24 or 96 h, as well.
While in the first case weaker signals are possible, in the second
THOMAS LIEHR and UWE CLAUSSEN
Comments
1. In this chapter only the preparation of peripheral blood metaphase
spreads is described. For more specific preparation protocols (e.g.
for bone marrow or solid tumors) see more specialized handbooks
(e.g. Wegner 1999).
2. It is a well-known fact that EDTA- or sodiumacetate-treated blood
samples can not be successfully cultured in short-term culture.
3. Sterile cell culture conditions have to be kept, so as not to contaminate
the short-term culture.
4. BrdU is a thymidine analogue which is readily incorporated into chromosomes. Cultures containing BrdU should be protected from light,
otherwise chromosome breaks will occur. Moreover, the yield of metaphase may be reduced compared to cultures without BrdU, as this
chemical is cytotoxic.
5. RNase and pepsin pretreatment conditions should be tested in each
laboratory on a single slide first. Both RNase and pepsin concentrations can be too stringent, resulting in clean slides without any remaining metaphase spreads or nuclei.
6. The pretreated slides can be hybridized immediately or stored at RT for
up to 3 weeks. If longer storage is necessary, slides are stable at -20°C
for several months.
7. Denaturation times of 2-3 min only will still allow the metaphase chromosomes to be evaluated. If only interphase nuclei are to be analyzed,
longer denaturation times of up to 6 min can lead to stronger and better estimable results.
8. In the current protocol the most common indirect probe-ligands, biotin and digoxigenine and one example of a detection system for them
are described. However, there are many other possibilities for directly
labeled ligands (like SpectrumGreen, SpectrumOrange, SpectrumAqua, etc.) or detection possibilities for the indirect ones (like CyS,
Cy5.5, TexasRed, etc.). The corresponding protocols for detection
are supplied by the companies and distributors and the chosen detection system mainly depends on the available fIlter sets of the own fluorescence microscope.
9. Incubation can be stopped - if necessary - after 24 or 96 h, as well.
While in the first case weaker signals are possible, in the second