74
THOMAS LIEHR and UWE CLAUSSEN
between meta- and anaphase (Ford and Hamerton 1956, Tjio and Levan
1956). The so called air-drying method for chromosome preparation of
Moorhead et al. (1956) includes in the following order, a hypotonic treatment with 0.075 M KCI (according to Hsu 1952, Hughes 1952), a so called
ftxation and several washing steps with Carnofs ftxative (= methanol/glacial acetic acid 3:1) and ftnally dropping of the suspension on a slide surface.
Even though such preparations of human chromosome have been
made for more than 40 years, their structure and the process of chromosome spreading are not completely understood up to now. Recent studies
have revealed that this spreading process is not based on a "bursting" procedure, as suggested for years, but that ftxed lymphocytes at the metaphase
stage spread very slowly on the slide (Hliscs et aL 1997). Moreover, this
surprisingly slow process leads to a spreading, which is humidity dependent - a fact described (Spurbeck et aI., 1996), but previously not well
understood. Moreover, chromosomes are elongated due to a stretching
or swelling process, arising during the metaphase spreading (Hliscs
et al. 1997, Claussen et al. 1999).
A standard preparation of human lymphocytes and a two-color FISH
approach including the possibility to enhance the DAPI-banding pattern
by BrdU (according to Heng and Tsui 1993) is described here.
Outline
1. Heparinized blood is added to cell culture medium, cell growth is stimulated by phytohaemagglutinin, and the short-term culture is incubated for 72 h at 37°C/5%CO z • For the last 16 h BrdU can be added to
the culture to enhance the DAPI banding pattern (see point 3). Cells are
harvested after the addition of co1cemid, subjected to a hypotonic
treatment, ftxed and washed in methanol/glacial acetic acid (3:1).
The resulting suspension is dropped on slides, which are air dried
and incubated over night at room temperature (see comment 1).
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 2-3 min at 75°C.
Biotinylated and/or digoxigenated probes are denatured for 5 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 leading to a green signal. The di-
THOMAS LIEHR and UWE CLAUSSEN
between meta- and anaphase (Ford and Hamerton 1956, Tjio and Levan
1956). The so called air-drying method for chromosome preparation of
Moorhead et al. (1956) includes in the following order, a hypotonic treatment with 0.075 M KCI (according to Hsu 1952, Hughes 1952), a so called
ftxation and several washing steps with Carnofs ftxative (= methanol/glacial acetic acid 3:1) and ftnally dropping of the suspension on a slide surface.
Even though such preparations of human chromosome have been
made for more than 40 years, their structure and the process of chromosome spreading are not completely understood up to now. Recent studies
have revealed that this spreading process is not based on a "bursting" procedure, as suggested for years, but that ftxed lymphocytes at the metaphase
stage spread very slowly on the slide (Hliscs et aL 1997). Moreover, this
surprisingly slow process leads to a spreading, which is humidity dependent - a fact described (Spurbeck et aI., 1996), but previously not well
understood. Moreover, chromosomes are elongated due to a stretching
or swelling process, arising during the metaphase spreading (Hliscs
et al. 1997, Claussen et al. 1999).
A standard preparation of human lymphocytes and a two-color FISH
approach including the possibility to enhance the DAPI-banding pattern
by BrdU (according to Heng and Tsui 1993) is described here.
Outline
1. Heparinized blood is added to cell culture medium, cell growth is stimulated by phytohaemagglutinin, and the short-term culture is incubated for 72 h at 37°C/5%CO z • For the last 16 h BrdU can be added to
the culture to enhance the DAPI banding pattern (see point 3). Cells are
harvested after the addition of co1cemid, subjected to a hypotonic
treatment, ftxed and washed in methanol/glacial acetic acid (3:1).
The resulting suspension is dropped on slides, which are air dried
and incubated over night at room temperature (see comment 1).
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 2-3 min at 75°C.
Biotinylated and/or digoxigenated probes are denatured for 5 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 leading to a green signal. The di-