Variation of signals in CTG method for different numbers of NIT-1 target cells and different incubation times. (A) NIT-1 cells were cultured in RPMI medium until 80-90% confluent and then detached with cell dissociation buffer to count for use in the assay. Cells at an initial density of 4 x 104 cells/well (50 μl) were titrated in 2-fold steps in a 100 μl volume per testing well. After overnight culture, the cells were washed, CTG reagent was added, and relative luminescence units (RLU) were measured at the various time points (A). The same procedure was used with lower NIT-1 cell numbers (B). The data shown are the mean and SD of triplicates and the results are representative of three independent experiments. For panel A, R2 is >0.98 for all curves (p < 0.0001); for panel B, R2 is >0.96 for all curves (p < 0.0001).