By measuring apoptosis, cell viability, and cytotoxicity in the same well, researchers are guaranteed that experimental conditions are identical for all measurements.By Sarah Shultz, Simon T.M. Allard, Paul Held, Jason Greene, and Peter Banks
 Figure 1. The ApoTox-Glo assay principle using the MultiTox-Fluor and Caspase-Glo 3/7 Assays. The MultiTox-Fluor Multiplex Cytotoxicity Assay measures the number of live and dead cells while the Caspase-Glo 3/7 Assay measures caspase-3/7 activities.
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Cell-based assays are a useful tool for early toxicology studies. Despite being a far simpler methodology than using animal models, cell-based assay workflows for toxicology applications can be laborious because assays are generally run in parallel microwell plates with multiple concentrations and treatment times, and can be costly with regard to use of cell culture consumables. Automation and multiplexing, where individual assays are combined in a single well, can simplify assay implementation, and provide more information because conditions are ensured to be the same for each assay. It also can reduce overall cell culture expenses. One such multiplexed assay application from
Promega Corporation simultaneously measures apoptosis, cell viability, and cytotoxicity. By measuring these three parameters in the same well, researchers are guaranteed that experimental conditions are identical for all measurements. This is important, for example, when one is working with primary cells where there is large donor variability and short supply.
Apoptosis is a major type of cell death, and is also used to describe a group of specific cell death mechanisms, although differentiated from necrosis (unintentional cell death), which involves lysis of cells and damage to the surrounding tissues. Activation of caspase-3/7 is a key biomarker of apoptosis. Cell viability is the determination of living or dead cells based on a total sample. This measurement provides information on how cells grow, act and react to external stimuli. Cytotoxicity characterizes the effects of an external compound, both positive and negative, on a cell. Data from a cytotoxicity assay that measures membrane integrity can either qualify or disqualify a compound from further studies.
Revealing compound effects on cells
The ApoTox-Glo cell-based assay (Figure 1) combines the Caspase-Glo 3/7 luminescent assay and MultiTox-Fluor fluorescent assay technologies (Promega Corporation) to measure viability, cytotoxicity, and caspase activation events in the same microplate well. These parameters are particularly useful to define mechanisms associated with a cytotoxic profile; all dispensing steps are automated using the Precision XS Automated Sample Processor (
BioTek Instruments, Inc.).
 Figure 2. The Precision XS Automated Sample Processor and Precision Power Software.
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The MultiTox-Fluor Assay is a single-reagent addition, homogeneous, fluorescent-based assay with non-lytic chemistry that allows measurement of live and dead cells in a single sample well. Specifically, for live cell assessment, live cell protease activity is measured by the fluorogenic, cell-permeant peptide substrate Gly-Phe-7-amino-4-trifluoromethyl coumarin (GF-AFC). This protease marker is active only in live cells and is inactivated if the cell membrane loses integrity. A second protease activity marker, the cell-impermeant peptide substrate bis-(Ala- Ala-Phe)-rhodamine 110 (bis-AAF-R110), is used to measure the activity of a dead-cell protease from cells that have lost membrane integrity and leaked the biomarker into the surrounding culture medium.
The Caspase-Glo 3/7 Assay, a homogeneous luminescent-based assay, measures caspase-3 and -7 activities in cell cultures as a marker of apoptosis. The assay provides a proluminescent caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD. This substrate is cleaved to release aminoluciferin, a substrate of luciferase used in the production of light. The amount of light produced correlates with caspase-3/7 activity.
Together, these assays provide three pieces of data per each sample well (cell viability, cytotoxicity, and caspase activity) to reveal compound affects on cells. The ApoTox-Glo assay also has a built-in control for assay normalization to help correct for well-to-well and day-to-day variability within a cell-based system.
Automation Instrumentation
A personal pipetting robot such as Biotek's Precision XS Automated Sample Processor (Figure 2) provides precise, automated control for a variety of applications including the ApoTox-Glo assay. Single- and multi-channel pipetting, bulk reagent dispensing and single channel aspirate/dispense are available to control all liquid handling steps of an assay including dilution of compounds, sterile dispensing and treatment of cells, and addition of assay reagents. Additionally, user-configurable multi-station platforms allow flexible experiment design: a wide range of sample tubes, microplates, and reagent vessels may be placed for optimal efficiency. The small footprint of the Precision XS enables sterile experimentation inside a biological safety cabinet, which is critical for any cell-based assay with treatment times beyond 12 hours.
When reading results of a multiplexed assay, a multi-mode microplate reader provides repeatability and speed compared to single-detection instruments. BioTek's Synergy 2 Multi-Mode Microplate Reader is used for measuring fluorescence at two wavelengths and luminescence in the ApoTox-Glo assay. The high-performance fluorescence detection system uses deep blocking filters (400Ex/505Em for the live-cell assay; 485Ex/520Em for the dead-cell assay) and dichroic mirrors for excellent performance in many research and screening applications such as the ApoTox-Glo assay. Additional detection modes for the modular system include fluorescence polarization, time-resolved fluorescence and absorbance.
 Figure 3. ApoTox-Glo assay results. A. (top) Assessment of Hep G2 cells after 2-hour treatment with bortezomib. B. (bottom) Assessment of Hep G2 cells after 4-hour treatment with staurosporine.
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Assay Protocol
Hep G2 cells were plated in 384-well format at a density of 5,000 cells in 10µL volumes of DMEM plus 10% fetal bovine serum and allowed to equilibrate for two hours. Staurosporine and bortezomib were twofold serially diluted and added to wells in 10µL volumes. Plates were incubated at 37°C in 5% CO
2 for 24 hours. MultiTox-Fluor Reagent was prepared as 10µL of each substrate in 2.5ml Assay Buffer, and 5µL was used per well. The contents of the microplate were mixed and incubated for 30 minutes at 37°C. Fluorescence measures of viability and cytotoxicity were read at two wavelengths on the Synergy 2 Multi-Mode Microplate Reader. Caspase-Glo 3/7 Reagent was then added in an additional 25µL volume, and luminescence was measured on the Synergy 2 Multi-Mode Microplate Reader after 30 minutes at room temperature.
ApoTox-Glo Assay Results
Proteolytic biomarkers for cell viability and cytotoxicity were measured using the MultiTox-Fluor Assay. Caspase-3/7 activity was then measured in the same well using the Caspase-Glo 3/7 Assay. As expected, both bortezomib (Figure 3A) and staurosporine (Figure 3B) compound treatments for 24 hours resulted in a dose-dependent decrease in viability, an increase in cytotoxicity, and an increase in caspase-3/7 activity consistent with apoptosis.
Summary
Multiplex assays provide a simple, efficient and cost-effective solution for simultaneous evaluation of compound effects on cells. When combined with automated instrumentation, these assay types, including the ApoTox-Glo assay, provide a small-scale, cost-effective cell-based screening method well-suited for
in vitro toxicology applications in academic labs, small biotechnology companies, and for secondary screening environments.n
arah Shultz, Senior Automated Solutions Scientist, and Simon T.M. Allard, Product Manager, are employees of Promega Corporation. Jason Greene, Product Manager, Paul Held, Senior Scientist, and Peter Banks, Scientific Director, are employees of BioTek Instruments, Inc.