To investigate the applicability of multiplexing CellTox™ Green with the portfolio of reporter assays that Promega offers, the compound terfenadine (200μM to 0.78μM) was chosen due to its known cytotoxic effects. Table 1 and Figure 2 show the results of all the reporter assays multiplexed with the CellTox™ Green Assay. There were no significant differences between the IC50 values determined with and without CellTox™ Green Dye present. There was a slight decrease in the overall luminescent signal due to the introduction of CellTox™ Green. A point of interest in these data was the NanoLuc® luciferase response to terfenadine. The NanoLuc® luminescent signal does not decrease as much the signal generated from the other luciferase reporters. The NanoLuc-PEST™ luciferase has a much more pronounced decrease in luminescent signal as the cells are killed. We believe that reduction in the fold-change in signal is due to the stability of the NanoLuc® luciferase. This evolved luciferase protein is more stable outside of the cell (3) . Therefore, once the cells are killed, the NanoLuc® luciferase will retain activity in the cell medium, reducing observed luciferase signal loss. This phenomenon illustrates the importance of carefully selecting the appropriate luciferase reporter for your experimental system.
Table 1. The IC50 values of the reporter assays are not significantly different in the presence or absence of CellTox™ Green Dye. The table shows the statistical 95% confidence intervals obtained for the cytotoxic agent terfenadine, calculated using GraphPad Prism® 5 software. The calculations are based on replicates of six.The IC50 values of the reporter assays are not significantly different in the presence or absence of CellTox™ Green Dye. The table shows the statistical 95% confidence intervals obtained for the cytotoxic agent terfenadine, calculated using GraphPad Prism® 5 software. The calculations are based on replicates of six.
The cells expressing Renilla luciferase showed a pronounced decrease in the luminescence curve and a correspondingly sharp increase in the fluorescence curve. To test if this was just a compound effect on this particular cell line, ionomycin was used to treat the cells instead of terfenadine. The ionomycin was diluted threefold from 100μM to 0.02μM. Figure 3 illustrates that the abnormal curves were not seen with the ionomycin experiment, and yet the IC50 values were still not significantly different. The multiplexed data show that cytotoxic events can reduce the reporter signal, and therefore, a cytotoxicity assay such as CellTox™ Green becomes essential for correct data interpretation.
Figure 2. Graphs of fluorescent and luminescent data from the reporter assays completed with the compound terfenadine.
The graphs reveal the inverse concordance that is expected from the multiplex of the CellTox™ Green Cytotoxicity Assay (fluorescence, RFU) and the luciferase reporter assay (luminescence, RLU). Luminescent signal declines as cells die and produce less protein. Fluorescent signal rises as the cell membrane becomes compromised, allowing the CellTox™ Green Dye to bind to the DNA. Panels A, B, and C are the Bright-Glo™, Steady-Glo®, and ONE-Glo™ Assays performed on HEK293 cells producing firefly luciferase constitutively; panel D is the Renilla-Glo™ assay performed on HEK293 cells producing Renilla luciferase constitutively; panels E and F are the Nano-Glo™ assays performed on HEK293 cells producing either NanoLuc™ luciferase or NanoLuc™-PEST luciferase constitutively, as indicated. Data are the average and standard deviation of six replicates.
Figure 3. IC50 values obtained for ionomycin.
The inverse concordance is shown again between the luminescent and fluorescent data. The luminescence is reduced due to the multiplex of the assays, but IC50 values are not significantly different. IC50 values are reported as 95% confidence intervals as calculated using GraphPad Prism® 5 software on replicates of six.