Figure 1 Two types of cytotoxicity assay. In the Cr-release assay, an increase in membrane permeability is measured. In the lUdR-release assay, disintegration of nuclear DNA is measured.

cell membrane is damaged. Thus, cell death can be determined by measuring radioactivity released from dead cells into the supernatant (Figure 1). Results are usually expressed as specific Cr release (%) calculated according to the following formula:

(experimental release) — (spontaneous release) ^ (maximum release) — (spontaneous release) '

where spontaneous release is the background radioactivity released in the absence of the effector and maximum release is the amount of released radioactivity when the target cells are maximally lysed by acid or detergent. The specific Cr release does not faithfully represent the actual percent of dead cells. This is because the efficiency of Cr uptake and spontaneous release of Cr can significantly vary from cell to cell in a heterogeneous cell population, and the maximum release induced by detergent is, for unknown reasons, usually significantly (approximately 20% on average) higher than Cr release when 100% of the cells are lysed by the effector. In order to quantitate the lytic activity of the effector, titration of the effector activity is necessary. Determination of lytic activity at a single effector: target ratio does not give an accurate estimate of cytotoxicity, as a small difference in the values of specific Cr release (less than 10%) is not reliable. Furthermore, above the plateau (79-80%), differences cannot be detected. Generally, the Cr release assay is used for cell-mediated target cell lysis because the presence of an excessive number of effector (or attacker) cells becomes problematic when using the dye exclusion assay. Furthermore, because of the ease of quantitation, this assay is favored when a large number of samples is involved.

For quantitative measurement of DNA disintegration, labeling nuclear DNA with l25IUdR or 3H-thymidine is widely used. Since labeling nuclear DNA depends on cellular synthesis of DNA, this method is applicable only for rapidly dividing cells such as tumor cells or blast cells. When a dividing population of cells are cultured in the presence of 12<iIUdR for 1-2 hours, a portion of cells incorporate the label into newly synthesized DNA strands. The chromosomal DNA remains in the nuclear matrix even after lysing cells with detergent. When nuclear DNA fragmentation takes place, small DNA fragments become released from the nucleus. In the assay, after incubation with the effectors, the whole cell mixtures are lysed with detergent and radioactivity in the supernatant is measured after pelleting nuclei by centrifugation. DNA fragmentation is usually expressed as percentage of specific IUdR release, which is calculated in a similar manner as the above-mentioned Cr release.

See also: Antibody-dependent cellular cytotoxicity; Apoptosis; Complement, membrane attack pathway; Cytotoxic T lymphocytes; Cytotoxicity, mechanisms of; Fas (CD95) and fas ligand; Granzymes; Lymphok-ine-activated killer (LAK) cells; Natural killer (NK) cells; Perforin; Tumor necrosis factor a; Lymphotox-in.

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