SubscribeToday’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.
Assay multiplexing for HTS
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 well1. This multiplexed format not only saves time and consumable cost, but also saves on 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 (figure 1). Table 1 highlights the assays used in this application.
Table 1: Cell-based assays for multiplexing applications
| Assay | Readout mode | Parameter measured |
| CellTiter-Glo® | Luminescence | Cell viability based on the quantification of ATP contained in viable cells in culture. Luminescence is directly proportional to the number of viable cells. |
| EnduRenTM | Luminescence | Renilla luciferase reporter luminescence via a protected coelenterazine substrate designed to generate Renilla luminescence from living cells. Once inside the cell, the protective groups of the substrate are cleaved by intracellular esterases, generating coelenterazine which reacts with Renilla to produce light. Peak luminescence is achieved after 1.5 hours of substrate addition to cells, and signal is stable for > 24 hours. |
| ViviRenTM | Luminescence | Renilla luciferase reporter luminescence via a protected coelenterazine substrate designed to generate Renilla luminescence from living cells. Once inside the cell, the protective groups of the substrate are cleaved by intracellular esterases, generating coelenterazine which reacts with Renilla to produce light. Peak luminescence is achieved after 2 minutes of substrate addition to cells, with signal half-life from 8 - 15 minutes. |
Fig. 1: BMG LABTECH's high-end multimode plate reader PHERAstar is used to perform multiplexed luciferase reporter and cell viability assays
Multiplexing Luciferase Reporter and Cell Viability Assays
Promega's luminescent CellTiter-Glo® assay2 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 37°C / 10% CO2. EnduRen™ Live Cell Substrate was added by the Equator to one plate of cells at a final concentration of 60 μM and allowed to incubate with the cells for 2 hours at 37°C / 10% CO2. 10 μM 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 μM, 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.
Correlation of the Renilla reporter gene signal with the overall cell number
To correlate Renilla luciferase reporter gene signal for cell viability, Promega's 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 LABTECH's PHERAstar in luminescence 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.
Fig. 2: Coupled Renilla reporter activity with cell viability. Renilla luminescence is first recorded with the PHERAstar. CellTiter-Glo® (CTG) Reagent is added, followed by luminescence reading. The kinetic profile of a Renilla reporter gene is correlated with the overall cell number.
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 μM 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.
Fig. 3: Coupled Renilla reporter activity with cell viability. Renilla luminescence is first recorded with the PHERAstar. CellTiter-Glo® (CTG) Reagent is added, followed by luminescence reading. Compared to EnduRen™, the ViviRen™ substrate produces higher overall luminescence signal. The kinetic profile of a Renilla reporter gene is correlated with the overall cell number.
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 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 LABTECH's PHERAstar to record luminescence from microplates of different well densities up to 1536-wells.
References
1) Farfan, A. et al.: (2004) Cell Notes 10, 2-5
2) Wesierska-Gadek, J. et al.: (2003) Cell Notes 6, 2-5
Contributing author:
Tracy Worzella
Promega Corporation, Madison, WI, USA