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Promegas Multiplexed Luciferase Reporter and Cell Viability Assays performed on the PHERAstar

lla luminescence and initiate ATP-dependent luminescence, which was recorded to measure the cell viability.


When making correlations between experimental conditions and the expression of a reporter gene, other events associated with cell physiology may affect reporter gene expression. Using live cell reporter substrates, it is possible to track the response of a Renilla reporter in real time by measuring luminescent Renilla reporter activity. Including a cell viability assay allows one to correlate reporter response with overall cell number. For this application, the Renilla reporter expression was optimal at 3 hours of treatment with 10 M isoproterenol in both assay formats tested, regardless of which substrate was used. Cell viability did not change over the time tested, indicating that changes in reporter response were due to the treatment and not changes in cell number. Using the same experimental conditions, the results also show the increased luminescence generated by the ViviRen substrate over the EnduRen substrate. The ViviRen substrate would be a good option when using cell lines that are weakly expressing Renilla luciferase, or for instances when fewer cells are being used as is the case for 1536-well format. Low volume 384 results also correlate with those in 1536, suggesting that miniaturization of these assays does not compromise data quality.


Conclusion
Multiplexed cell-based assays allow for multiple parameters to be measured within the same well. With reporter assays, the expression of a luciferase reporter can be greatly affected by the overall health of the cell. By multiplexing a reporter a
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