Tracy Worzella and Brad Larson
Promega Corporation, Madison, WI, USA
Application Note 139 Rev. 7/2006
Today's high-throughput screening facilities face increasing demands to generate more information from their existing compound libraries. One method of obtaining this information is to run assays sequentially, looking at one parameter followed by another in different plates. While this option may produce the desired data, the increased time and consumable costs are drawbacks. A more appealing method for data generation is to perform assays in a multiplexed format in which several parameters can be measured within the same well. This multiplexed format not only saves time and consumable cost, but also saves on the useage of valuable test compounds.
This concept of assay multiplexing is demonstrated here using several cell-based assays multiplexed together. There are inherent properties to cell assays that make them attractive for multiplexed cell-based applications. Cell-based assays are especially vulnerable to variations due to differences in cell growth and metabolism that can arise from plate-to-plate. Cell culture itself is also expensive. By multiplexing assays, fewer cells are needed to acquire the same amount of data. Using the same cells for subsequent assays can also ensure more precise data. In this application note, we demonstrate the combination of several Promega cell-based assays multiplexed in both low-volume 384- and 1536-well plate formats. The BMG LABTECH PHERAstar microplate reader is used to record luminescence. Table 1 highlights the assays used in this application.
Materials and Methods
Multiplexing Luciferase Reporter and Cell Viability Assays
Promegas luminescent CellTiter-Glo assay was multiplexed with either the luminescent EnduRen Live Cell Substrate, or the luminescent ViviRen Live Cell Substrate. The experimental set-up was similar for each assay combination.
For the low-volume 384 assay format, a density of 12,000 stably transfected HEK 293 cells per well was plated with the Deerac Fluidics Equator. Cells were allowed to attach for 10 hours at 37C / 10% CO2. EnduRen Live Cell Substrate was added by the Equator to one plate of cells at a final concentration of 60 M and allowed to incubate with the cells for 2 hours at 37C / 10% CO2. 10 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 Renilla 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.
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 assay with a cell viability assay, it is possible to determine if reporter response variations are due to changes in cell number and health. Depending on the Renilla expression levels or the number of cells being used, the EnduRen and ViviRen Live Cell Substrates provide options for the customer looking to observe real-time reporter kinetics. The sensitivity of the PHERAstar allowed for luminescence detection from both reporter substrates. The data generated here also showcases the ability of BMG LABTECHs PHERAstar to record luminescence from microplates of different well densities up to 1536-wells.
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