High-Sensitivity Cytotoxicity Assays for Nonadherent Cells

High-sensitivity cytotoxicity assays refer to assays that can detect high levels of cell kill, to many powers of 10, and that can detect, ideally, a single remaining viable cell. Two such assays are described here, which have been used with Raji B-lymphoma cells, and are applicable to other nonadherent target cells. The first is a cell-counting assay, performed over a 3-wk period, which provides a simple, reliable, and sensitive assay of cytotoxicity. By determining the time required for 16-fold multiplication, the apparent fraction surviving can be calculated. This assay does not correct for treatment-induced delays in cell division and is dependent on maintaining the cells in exponential growth. The second assay measures colony-forming units using a limiting dilution method. Feeder cells are required to obtain a high cloning efficiency. Each dilution is plated in 48 wells of a 96-well plate, and positive wells are scored rapidly, by eye, after two wk.