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