Chapter 33
Identification of Cell Lines
Discrimination between and identification of cell lines are important in
order to avoid confusion due to mislabeling and accidental cellular contamination. Cell morphology is not a reliable criterion for the identification of cells, because the shape of a cell changes easily with different
culture conditions, and a cell population usually consists of variousshaped cells, even in a cloned cell line. Chromosome analysis can be used
to discriminate between and identify cell lines; however, lepidopteran
cell lines have numerous small chromosomes and this makes determination of the karyotype difficult in these cases. Isozyme pattern analysis
is also useful in the identification of cell lines, but isozyme pattern analysis
does not clearly distinguish cell lines derived from taxonomically close
species, or cell lines derived from the same species. A reliable method for
discriminating cell lines is the recently developed restriction fragment
length polymorphism (RFLP). However, this method has some drawbacks: radioisotopes, a large number of cells, and southern hybridization are needed in RFLP, and the procedure itself is not simple. More
recently, a polymerase chain reaction (PCR) method for the study of
phylogeny came into use. This technique is a good tool for the discrimination of cell lines. Furthermore, a method of random amplified polymorphic DNA (RAPD)-PCR has been developed. Because gene analyses
of insect cell lines are scarce, a method of RAPD-PCR using appropriate
short single primers seems to be most practical.
Subprotocol 1
Isozyme Analysis by Agar Gel Electrophoresis
This is a technique that can be used for distinguishing cell lines derived
from different species. However, discrimination of cell lines from the same
species, even from different organs or tissues, is not usually possible using this method. Isozyme analysis by agar gel electrophoresis consists of
two stages: first, separation of protein (enzyme) by electrophoresis, and,
second, staining the separated proteins with a coupler-containing specific enzyme substrate. In the first step, agarose gel or natural polyacrylamide gel electrophoresis can be used. In addition, isoelectric focusing
and two-dimensional electrophoresis may be applied.
In the second step, selected enzyme activity is checked; for example,
the activity of malic enzyme, isocitric dehydrogenase, phosphoglucose
isomerase, or phosphoglucomutase is often examined for insect cells. The
PROTOCOL
Identification of Cell Lines
Discrimination between and identification of cell lines are important in
order to avoid confusion due to mislabeling and accidental cellular contamination. Cell morphology is not a reliable criterion for the identification of cells, because the shape of a cell changes easily with different
culture conditions, and a cell population usually consists of variousshaped cells, even in a cloned cell line. Chromosome analysis can be used
to discriminate between and identify cell lines; however, lepidopteran
cell lines have numerous small chromosomes and this makes determination of the karyotype difficult in these cases. Isozyme pattern analysis
is also useful in the identification of cell lines, but isozyme pattern analysis
does not clearly distinguish cell lines derived from taxonomically close
species, or cell lines derived from the same species. A reliable method for
discriminating cell lines is the recently developed restriction fragment
length polymorphism (RFLP). However, this method has some drawbacks: radioisotopes, a large number of cells, and southern hybridization are needed in RFLP, and the procedure itself is not simple. More
recently, a polymerase chain reaction (PCR) method for the study of
phylogeny came into use. This technique is a good tool for the discrimination of cell lines. Furthermore, a method of random amplified polymorphic DNA (RAPD)-PCR has been developed. Because gene analyses
of insect cell lines are scarce, a method of RAPD-PCR using appropriate
short single primers seems to be most practical.
Subprotocol 1
Isozyme Analysis by Agar Gel Electrophoresis
This is a technique that can be used for distinguishing cell lines derived
from different species. However, discrimination of cell lines from the same
species, even from different organs or tissues, is not usually possible using this method. Isozyme analysis by agar gel electrophoresis consists of
two stages: first, separation of protein (enzyme) by electrophoresis, and,
second, staining the separated proteins with a coupler-containing specific enzyme substrate. In the first step, agarose gel or natural polyacrylamide gel electrophoresis can be used. In addition, isoelectric focusing
and two-dimensional electrophoresis may be applied.
In the second step, selected enzyme activity is checked; for example,
the activity of malic enzyme, isocitric dehydrogenase, phosphoglucose
isomerase, or phosphoglucomutase is often examined for insect cells. The
PROTOCOL