DNA Analysis: DNA content can be measured by propidium iodide fluorescence with a CCD camera or flow cytometry, although the generation of the necessary single-cell suspension will, of course, destroy the topography of the specimen. DNA can be estimated in homogenates with Hoechst 33258 and other DNA fluorochromes such as DAPI, propidium iodide, or Pico Green (Molecular Probes). Analysis of DNA content is particularly useful in the characterization of transformed cells that are often aneuploid and heteroploid.

DNA Hybridization: Hybridization of specific molecular probes to unique DNA sequences (Southern blotting) can provide information about species specific regions, amplified regions of the DNA, or altered base sequences that are characteristic to that cell line. Thus strain-specific gene amplifications, such as amplification of the dihydrofolate reductase (DHFR) gene, may be detected in cell lines selected for resistance to methotrexate; amplification of the MDR gene in vinblastine-resistant cells overexpression of a specific oncogene, or oncogenes in transformed cell lines or deletion, or loss, of heterozygosity in suppressor genes. Although DNA aberrations can be detected in restriction digests of extracts of whole DNA, this is limited by the amount of DNA required.

It is more common to use the polymerase chain reaction (PCR) with a primer specific to the sequence of interest, enabling detection in relatively small numbers of cells. Alternatively, specific probes can be used to detect specific DNA sequences by in situ hybridization having the advantage of displaying topographical differences and heterogeneity within a cell population.

DNA fingerprinting: DNA fingerprints appear to be quite stable in culture, and cell lines from the same origin, but maintained separately in different laboratories for many years, still retain the same or very similar DNA fingerprints. DNA fingerprinting is a very powerful tool in determining the origin of a cell line, if the original cell line, or DNA from it or from the donor individual, has been retained. This emphasizes the need to retain a blood, tissue, or DNA sample when tissue is isolated for primary culture. Furthermore, if a cross-contamination or misidentification is suspected, this can be investigated by fingerprinting the cells and all potential contaminant.

Antigenic Markers: Immunostaining and ELISA assays are among the most useful techniques available for cell line characterization facilitated by the abundance of antibodies and kits which is commercially available. Antibody is essential to be certain of its specificity by using appropriate control material. This is true for monoclonal antibodies and polyclonal antisera alike; a monoclonal antibody is highly specific for a particular epitope.

Immunostaining: Antibody localization is determined by fluorescence, wherein the antibody is conjugated to a fluorochrome, such as fluorescein or rhodamine, or by the deposition of a precipitated product from the activity of horseradish peroxidase or alkaline phosphatase conjugated to the antibody. Various methods have been used to enhance the sensitivity of detection of these methods, particularly the peroxidase linked methods. In the peroxidase–anti-peroxidase (PAP) technique, a further amplifying tier is added by reaction with peroxidase conjugated to anti-peroxidase antibody from the same species as the primary antibody. Even greater sensitivity has been obtained by using a biotin-conjugated second antibody with streptavidin conjugated to peroxidase or alkaline phosphatase or gold-conjugated second antibody with subsequent silver intensification.

Differentiation: Many of the characteristics described under antigenic markers or enzyme activities may also be regarded as markers of differentiation, and as such they can help to correlate cell lines with their tissue of origin as well as define their phenotypic status. Although sometimes constitutively expressed (e.g., melanin in B16 melanoma or Factor VIII in endothelial cells), expression of differentiated lineage markers may need to be induced before detection is possible.

 

Interesting facts:

• Trypsin cuts the adhesion proteins in cell-cell and cell-matrix interactions
• EDTA, a chelator of divalent cations, is added to trypsin solutions to enhance activity.
• The calcium and magnesium in the extracellular matrix, which aids in cell-cell adhesion, also obliterates the peptide bonds that trypsin acts on.
• The EDTA is added to remove the calcium and magnesium from the cell surface which allows trypsin to hydrolyze specific peptide bonds. This activity can be arrested by adding a serum media mixture or a trypsin inhibitor (from soybean, for example) in serum-free systems.