Cytotoxicity Assays

Nobukata Shinohara, Mitsubishi-Kasei Institute of Life Science, Tokyo, Japan

A variety of immunological effectors attack cells, resulting in cytolysis of the targets. These include specific antibody with complement, cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, lympho-kine-activated killer (LAK) cells, the effectors of antibody-dependent cellular cytotoxicity (ADCC), macrophages, neutrophils and certain substances secreted by cells such as tumor necrosis factor (TNF) and perforin. CTLs and NK cells are known to utilize dual pathways of cytotoxic machinery: perforin + granzyme mediated and Fas ligand mediated. Cell death is generally categorized into two different types. One, called programmed cell death (apoptosis), involves active metabolic process of the dying cell resulting in disintegration of nuclear DNA and an increase in the permeability of the cell membrane. The other type, called necrosis, does not involve active metabolic process and results in an increase in membrane permeability without immediate nuclear disintegration. Cell-mediated cell lysis generally induces the former type of cell death, whereas cell lysis mediated by antibody and complement causes the latter type. Accordingly, there are two different principles for detecting cell death: one for measurement of increased membrane permeability and the other for detecting DNA disintegration.

Two types of methods for monitoring membrane permeability of cells are widely used as cytotoxicity assays. One is the dye exclusion test. This assay-involves microscopical counting of dead cells stained with a dye, such as trypan blue, to which viable cell membrane is impermeable. These agents, because of their basic nature, stain cell nuclei when they have entered the cells across the damaged cell membrane. By this method, the percentage of dead cells can be directly determined. The other method is the chromium-release assay, in which target cells arc labeled with radioactive 5'Cr. When cultured in the presence of a trace amount of Na2CrO.,, viable cells take up Cr6f. Once inside the cell, the Crh~ is reduced to Cr'~. The Cr'1 binds to multivalent anions, including proteins present in the cells. This results in the formation of molecular complexes which are not easily released from cells unless the

Cr-release assay

(membrane damage)

lUdR assay

(DNA fragmentation)

12SIUdR

Na?i1CrOi

Effector cells i3

lUdR assay

(DNA fragmentation)

12SIUdR

Effector cells

37TC 1 hour Washing

+ Triton X

37TC 1 hour Washing

+ Triton X

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