( Tracy Worzella and Brad Larso...)Promegas Multiplexed Cell Viability and Apoptosis Assays performed on the PHERAstar

Tracy Worzella and Brad Larson
Promega Corporation, Madison, WI, USA

Application Note 138 Rev. 07/2006

Correlated measurement of an apoptosis marker (or caspase activity) and cell viability

Monitoring of both luminescence and fluorescence output signals from one assay well

Assay miniaturization up to 1536-well format

Introduction
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 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 both luminescence and fluorescence, depending on the multiplex combination. Table 1 highlights the assays used in this application.

Materials and Methods

Jurkat cells

Anti-FAS monoclonal antibody

Corning Low-volume 384-well plates, catalog no. 3673

Corning 1536-well plates, catalog no. 3937

Promega Caspase-Glo 3/7 Assay System, catalog no. G8091*

Promega CellTiter-Blue Cell Viability Assay, catalog no. G8080

Promega Apo-ONE Homogeneous Caspase 3/7 Assay, catalog no. G7790

Deerac Fluidics Equator HTS low-volume liquid dispenser

BMG LABTECH PHERAstar (figure 1)

BMG LABTECH PHERAstar luminescence optic module

BMG LABTECH PHERAstar fluorescence optic module (Ex: 485 nm; Em: 520 nm)

BMG LABTECH PHERAstar fluorescence optic modules (Ex: 540 nm; Em: 590 nm)

Multiplexing Cell Viability and Apoptosis Assays
Promegas fluorescent CellTiter-Blue cell viability assay was multiplexed with either the luminescent Caspase-Glo 3/7 assay, or the fluorescent Apo-ONE assay. The experimental set-up was similar for each assay combination.

For the low-volume 384 assay format, a density of 10,000 Jurkat cells per well was plated with the Deerac Fluidics Equator. Next, a range of anti-FAS monoclonal antibody was added to the plate, with the final concentration per well ranging from 400 ng/mL down to 0 ng/mL of antibody. The plates were then incubated at 37C / 5% CO₂ for a total of 5 hours to induce apoptosis. 3 hours into the 5 hour treatment, CellTiter-Blue reagent was added to each well with the Equator (note: for plates later receiving Caspase-Glo 3/7 reagent, CellTiter-Blue was diluted 1:4 in 1X PBS before addition to the assay plate). After the CellTiter-Blue addition, plates were incubated for 2 hours at 37C / 5% CO₂. When the 5 hour incubation with anti-FAS antibody was complete, fluorescence was recorded at excitation 540 nm and emission of 590 nm with the PHERAstar. The caspase reagents were then added to the plates with the Equator. Apo-ONE was added to one plate containing CellTiter-Blue reagent, and the plate was incubated at room temperature for 1 hour, followed by fluorescence reading with the PHERAstar at excitation 485 nm and emission of 520 nm. Caspase-Glo reagent was added to the plate receiving the diluted CellTiter-Blue reagent, and incubated for 1 hour at room temperature. Luminescence was then recorded with the PHERAstar.

For the 1536-well assay format, a density of 4,000 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
Promegas CellTiter-Blue assay multiplexed with either Caspase-Glo 3/7 (figure 2) or Apo-ONE assay was prepared in both low-volume 384- and 1536-well format (figure 3). Cell viability and apoptosis were sequentially measured with BMG LABTECHs PHERAstar in both luminescence and fluorescence modes, depending on the multiplex combination. Jurkat cells were treated with various concentrations of anti-FAS monoclonal antibody for 5 hours to induce apoptosis. Cell viability was determined by adding CellTiter-Blue reagent to each well after drug addition and incubating for two hours before recording fluorescence (Ex: 540 nm; Em: 590 nm). The caspase activity was then measured by adding either Caspase-Glo 3/7 or Apo-ONE reagent and incubating for an additional hour prior to recording luminescence or fluorescence (Ex: 485 nm; Em: 520 nm) respectively.

Figures 2 and 3 show the results of two experiments to determine the method of cell death caused by different concentrations of anti-FAS antibody in Jurkat cells. The two experiments measured two different endpoints: reduction of a resazurin dye as an indicator of viable cells and caspase activity as a marker for apoptotic cells. The data show that with increasing concentration of anti-FAS antibody, an increase in caspase-3/7 activity, with a corresponding decrease in cell viability, is observed.

The results suggest that the cell population studied is less viable over the range of treatment due to an increase in apoptosis, as opposed to necrosis.For all cell viability and apoptosis multiplexing combinations, results in 1536 format are comparible to results in 384-well format.

Conclusion
Each of the experiments shown here highlights the ability to perform different assays within the same assay well. Using a non-lytic assay first, such as the CellTiter-Blue assay used here, allows for the sequential multiplexing of several different reagents within the same well. Multiplexed cell-based assays allow for multiple parameters to be measured within the same well. One example of this is to determine the method of cell death following a certain treatment protocol. By performing two assays within the same well, information on mode of action of drugs could be obtained faster and with less consumable usage. Data from miniaturized assays in 1536-well format are comparable to those run in low volume 384, indicating that smaller assay volume does not compromise the results obtained in higher density formats. The data generated here also showcases the ability of BMG LABTECHs multifunctional PHERAstar to record multiple output signals from the same assay well.

* See Promegas website for patent marking information
www.promega.com

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