Based on the observation that STAT1-CC-expressing cells show increased activity of the endogenous ISRE promoter element [13] , we established cell lines that stably expressed an ISRE-containing gene promoter driving a click beetle luciferase reporter gene CBG99luc (Figure 2A ). We used 2fTGH cells (the parental line for STAT1-deficient U3A cells) as well as HEK293T cells that both express endogenous STAT1. Clonal lines showing IFN-b-inducible ISRE-promoter driven luciferase activity were designated 2fTGH-or HEK293T-ISRE-CBG99 cells. For initial assay development, we used the 2fTGH-and HEK293T-ISRE-CBG99 cells to establish an optimal luciferase light reaction time for both cell-lines. Based on luminescence signal stability over the time course of the reaction, an optimal readout window of 40-70 min after the start of the reaction was chosen for subsequent experiments ( Figure 2B ). After optimization of cell growth time, response to various IFN-b treatment times and concentrations were tested in 2fTGH and HEK293T cell lines. Each cell line exhibited a distinct IFN-b treatment time for maximal signal: 7-12 h for 2fTGH-ISRE-CBG99 cells and 14-24 h for HEK293T-ISRE-CBG99 cells. Although a lower signal magnitude was obtained with 2fTGH-ISRE-CBG99 cells compared to HEK293T-ISRE-CBG99 cells, the 2fTGH-ISRE-CBG99 cells show more specificity for IFN-b (compared to IFN-c) treatment at all IFN-b treatment time periods tested ( Figure 2E ) and over a range of IFN-b (and IFN-c) concentrations ( Figure 2F , G). Thus, the 2fTGH-ISRE-CBG99 cells were chosen for further assay development.