38] Assay of a cytocidal protein excreted by activated macrophages

Abstract

This chapter describes two assays of cytocidal protein excreted by activated macrophages. Macrophages, when activated by a variety of agents, become cytotoxic for nucleated target cells. One way macrophages mediate the killing of the target cells is through the release of a soluble factor designated as macrophage cytotoxic factor (M ϕ -CF). When cells, which adhere to the surface of culture vessels, are used as targets, the ability of M ϕ -CF to kill the target cells can be measured by determining the number of dead cells that detach from the culture vessel. Another assay method involves the use of radiolabeled thymidine or its analog, iododeoxyuridine, because target cells damaged by M ϕ -CF cease to incorporate these DNA precursors. Medium containing M ϕ -CF can be generated by culturing mouse, rat, guinea pig, or rabbit macrophages in the presence of an activator. Tissue culture medium (TCM) is used to maintain all of the target cell lines and is also used in the M ϕ -CF assays.