Chapter 32
Karyotype Analysis
The karyotype is one of the characteristics of a cell line. However, it is
not necessarily the same as that of cells in vivo. During culture, the karyotype of cells often changes. Consequently, many continuous cell lines are
polyploid or heteroploid. Usually, the chromosome number is distributed over a wide range. This pattern of distribution may change with
passages. Therefore, the karyotype is one characteristic of a continuous
cell line, but discrimination of cell lines according to karyotype is not
possible, especially among cell lines derived from taxonomically close
species. Nevertheless, the determination of karyotype is a basic requirement for describing continuous cell lines.
Materials
- Biologic microscope
- Centrifuge and centrifuge tubes
- Pasteur pipettes
- Petri dishes
- Cover glass
- Slide glass
- 5 X 10- 3 M colchicine solution
- Hypotonic KCI solution (the concentration should be predetermined
by examining the preparation made with different concentration of
KCI. If cell membrane remains intact the concentration is too high. If
only nuclei are visble, the concentration is to low. If the concentration
is suitable, cell membrane is broken, cytoplasm scatters around nuclei, and chromosomes are discernible in the scattered cytoplasm of
the cells at mitotic metaphase; e.g. 0.58 % for mosquito cells)
- Carnoy's fixative (methanol: acetic acid ratio 3 : 1)
- Methanol
- Giemsa stain
- Canada balsam
Procedure
1. Passage a culture and wait until the cells enter the exponential growth
phase.
2. Add a 1/10 volume of colchicine solution to the culture.
PROTOCOL
Equipment
Solutions
Chemicals
Metaphase
arrest
Karyotype Analysis
The karyotype is one of the characteristics of a cell line. However, it is
not necessarily the same as that of cells in vivo. During culture, the karyotype of cells often changes. Consequently, many continuous cell lines are
polyploid or heteroploid. Usually, the chromosome number is distributed over a wide range. This pattern of distribution may change with
passages. Therefore, the karyotype is one characteristic of a continuous
cell line, but discrimination of cell lines according to karyotype is not
possible, especially among cell lines derived from taxonomically close
species. Nevertheless, the determination of karyotype is a basic requirement for describing continuous cell lines.
Materials
- Biologic microscope
- Centrifuge and centrifuge tubes
- Pasteur pipettes
- Petri dishes
- Cover glass
- Slide glass
- 5 X 10- 3 M colchicine solution
- Hypotonic KCI solution (the concentration should be predetermined
by examining the preparation made with different concentration of
KCI. If cell membrane remains intact the concentration is too high. If
only nuclei are visble, the concentration is to low. If the concentration
is suitable, cell membrane is broken, cytoplasm scatters around nuclei, and chromosomes are discernible in the scattered cytoplasm of
the cells at mitotic metaphase; e.g. 0.58 % for mosquito cells)
- Carnoy's fixative (methanol: acetic acid ratio 3 : 1)
- Methanol
- Giemsa stain
- Canada balsam
Procedure
1. Passage a culture and wait until the cells enter the exponential growth
phase.
2. Add a 1/10 volume of colchicine solution to the culture.
PROTOCOL
Equipment
Solutions
Chemicals
Metaphase
arrest