( M of isoproterenol was adde...)Promegas Multiplexed Luciferase Reporter and Cell Viability Assays performed on the PHERAstar

M of isoproterenol was added to all test plates to induce CRE. Induction was monitored at 0, 1-, 2-, 3-, and 5-hour time points. At each time point, ViviRen substrate was added by the Equator at a final concentration of 60 M, luminescence reporter signal was recorded, followed by addition of the CellTiter-Glo reagent by the Equator. Luminescence was recorded a second time to measure ATP content and cell number.

For the 1536-well assay format, a density of 4,000 stably transfected HEK 293 cells per well was plated with the Deerac Fluidics Equator. The remaining multiplex protocols were performed identically to the low-volume 384 protocols listed above.

Results and Discussion
To correlate Renilla luciferase reporter gene signal for cell viability, Promegas CellTiter-Glo assay was multiplexed with either the EnduRen Live Cell Substrate or the ViviRen Live Cell Substrate. Both multiplexed assay combinations were prepared in both low-volume 384- and 1536-well format and Renilla expression and cell viability were sequentially measured with BMG LABTECHs PHERAstar in luminsecence mode (figure 2 and figure 3).

HEK 293 cells were treated with isoproterenol for 5 hours to induce Renilla reporter expression and a correlated kinetic profile of the reporter signal and cell viability were recorded over this time. The EnduRen Live Cell Substrate was added 2 hours before CRE induction and allowed to incubate with the cells. The ViviRen Live Cell Substrate was added directly before each measurement point. After luminescence reporter signal determination, the CellTiter-Glo reagent was added at each measurement point to inactivate Reni
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