Frequency Determination of Killer Cells by a Single-Cell Cytotoxic Assay

This chapter discusses the frequency determination of killer cells by a single-cell cytotoxic assay. The development of the single-cell assay has provided a direct approach to estimate the frequency of cytotoxic cells present in heterogeneous cell populations. The most commonly used single-cell assay is based on the ability of the cytotoxic cell to bind a corresponding target, resulting in an effector cell-target–cell conjugate. These conjugates are dispersed in a thin layer of agarose and incubated at 37° until target lysis takes place. Single-target cell lysis is determined microscopically by trypan blue uptake into lymphocyte-bound target cells. The fraction of lymphocytes bound to dead target cells is enumerated, and the frequency of cytotoxic cells in the test sample is calculated. The single-cell assay is simple to perform, highly reliable, and permits the enumeration of large numbers of conjugates. It measures the frequency of cytotoxic cells, and thus several properties of these cells, such as rate of lysis, heterogeneity, and surface markers, can be examined. It is applicable to most cytotoxic systems. The assay can be easily performed in a few hours with minimal requirement for equipment.