Introduction
Cell-based assays are useful tools to investigate in vitro responses to a variety of target compounds and cell-signaling molecules. Using compatible assays capable of measuring multiple parameters in a single well (i.e., multiplexing) with the same population of cells is often desirable, particularly in secondary screening, to generate more biologically relevant data. Multiplexing assays also helps to provide normalized controls, save materials and reduce the time- and labor-intensive tasks of performing each assay individually.
By combining three assays into one multiplex assay, the ApoTox-Glo™ Triplex Assay provides a broader picture of what is occurring inside cells, specifically information about cell health, toxicity, and apoptosis events.
Here, we introduce a new multiplexing cell-based assay product called the ApoTox-Glo™ Triplex Assay. This triplex assay is intended for same-well measurement of three parameters: cell viability, cytotoxicity, and caspase activation events. The chemistry combines one luminescent and two fluorescent technologies into one convenient kit for users. Each of the three subsets of the assay also are available individually (CellTiter-Fluor™, CytoTox-Fluor™, and Caspase-Glo® 3/7), allowing users to optimize individual parameters of the assay.
By combining three assays into one multiplex assay, the ApoTox-Glo™ Triplex Assay provides a broader picture of what is occurring inside cells, specifically information about cell health, toxicity, and apoptosis events. Together these parameters help better define mechanisms associated with a cytotoxic profile. The assay is especially well suited for in vitro toxicology applications in academic labs, small biotechnology companies, and for secondary screening environments.
How the Assay Chemistry Works
The ApoTox-Glo™ Triplex Assay combines three existing Promega assay chemistries to assess viability, cytotoxicity and caspase activation events within a single assay well. The assay workflow occurs in two parts. The first part of the assay measures two protease biomarker activities (Figure 1); one is a marker of cell viability (live-cell), and the second is a marker of cytotoxicity (dead-cell). The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant substrate called GF-AFC. GF-AFC enters intact cells and is cleaved by the live-cell protease. Upon cleavage, a fluorescent signal is generated, which is proportional to the number of living cells. This live-cell protease becomes inactive upon loss of cell membrane integrity and leakage into the surrounding culture medium. A second, fluorogenic substrate called bis-AAF-R110 is used to measure dead-cell protease activity. Bis-AAF-R110, however, is cell-impermeant. Dead-cell proteases are released from cells that have lost membrane integrity. Because bis-AAF-R110 is not cell-permeant, essentially no signal from this substrate is generated by viable cells.
The live- and dead-cell proteases each produce a different fluorescent product, AFC and R110, respectively. These products have different excitation and emission spectra (Ex 400nm/Em 505nm and Ex 485nm/Em 520nm, respectively), allowing them to be detected simultaneously using a fluorescent plate reader.
The second part of the ApoTox-Glo™ Triplex Assay detects caspase-3/7 activity using a luminogenic, caspase-3/7 substrate that contains the tetrapeptide sequence DEVD in a reagent optimized for caspase activity, luciferase activity and cell lysis (Figure 2). Adding the caspase-3/7 substrate reagent, which contains the Ultra-Glo™ rLuciferase, results in cell lysis, intracellular caspase cleavage of the substrate and release of luciferin. The luciferin generates a luminescent signal produced by luciferase (1)
. The luminescent signal is proportional to the amount of caspase activity present and can be measured using a luminescent plate reader.
How to Perform the Assay
The ApoTox-Glo™ Triplex Assay protocol uses a simple sequential “add-mix-read” format (Figure 3). The volumes of each assay component can be scaled to meet the varying throughput needs of researchers, and this assay is amenable to automation. Table 1 provides a description of the recommended volumes used at each assay step.
Figure 3. Overview of the ApoTox-Glo™ Triplex Assay protocol. 
Table 1. ApoTox-Glo™ Triplex Assay protocol. 
How the Assay Performs
Cell viability, cytotoxicity, and caspase-3/7 activity were assessed using the ApoTox-Glo™ Triplex Assay with several different cell lines (K562, Jurkat, L929, and HepG2) treated for different periods of time with mechanistically different cytotoxic compounds (ionomycin, staurosporine, bortezomib, and SAHA) in 96-, 384-, and 1536-well assay plate formats. The cytotoxic profiles generated using the ApoTox-Glo™ Triplex Assay measurements demonstrate the expected trends for each compound's effect on the cells. In vitro cytotoxicity is dependent upon compound dosage and exposure period. The kinetics of measurable cytotoxic biomarkers can vary widely between individual compounds and treatments. Therefore, it is important to characterize new compounds using multiple exposure periods(2)
.
Ionomycin was selected because it causes primary necrosis in less than five hours, typically without caspase activation, and is a commonly used cytotoxic agent. Ionomycin is a bacterial ionophore used in research to affect calcium transport across cell membranes. Specifically, ionomycin stimulates production of inflammatory cytokines. Ionomycin treatment of in vitro cells for approximately 4–6 hours typically results in a dose-dependent decrease in cell viability and an increase in cytotoxicity without caspase-3/7 activation. This type of cellular profile is consistent with primary necrosis (Figure 4).
Similar to ionomycin, staurosporine also is considered a standard cytotoxic agent. Specifically, staurosporine is an antibiotic and pleiotropic ATP-competitive kinase inhibitor that causes caspase activation in cells after approximately 6–8 hours. Staurosporine acts to inhibit protein kinases by preventing the binding of ATP. Staurosporine treatment of in vitro cells for approximately 4–6 hours typically results in a dose-dependent decrease in cell viability, an increase in cytotoxicity, and an increase in caspase-3/7 activity consistent with apoptosis (Figure 5).
Bortezomib or Velcade™ (Millennium Pharmaceuticals) is a proteasome inhibitor that is a new therapeutic treatment for multiple myeloma cancer. Bortezomib has been shown to inhibit proteasomes, cellular enzymes that play a role in regulation and cell growth processes, which in turn can lead to cancer cell death. Bortezomib treatment of in vitro cells for approximately 24 hours results similarly to staurosporine in a dose-dependent decrease in viability, an increase in cytotoxicity, and an increase in caspase-3/7 activity consistent with apoptosis (Figure 6).
Finally, suberoylanilide hydroxamic acid (SAHA) or orinostat, rINN, or Zolinza™ is a new anticancer therapeutic that has been shown to inhibit histone deacetylases (HDAC). SAHA treatment of in vitro cells for approximately 24–48 hours results in a dose-dependent decrease in cell viability, increase in cytotoxicity, and an increase in caspase activation events, consistent with apoptosis (Figure 7).
Figure 7. ApoTox-Glo™ Triplex Assay results for suspension Jurkat cells treated with SAHA.
SAHA treatment of Jurkat cells for 24 hours results in a dose-dependent decrease in viability, increase in cytotoxicity, and an increase in caspase-3/7 activity consistent with apoptosis.
Summary
Multiplexing with the Promega ApoTox-Glo™ Triplex Assay helps to provide researchers with information about cell health, toxicity and apoptosis events, while minimizing the assay workflow time. The unique combination of chemistry successfully provides data assessing cell viability, cytotoxicity, and caspase activation events occurring inside the same population of cells. The assay is well suited for multi-well plate formats (96, 384, and 1536), and serves as an especially useful tool for academic researchers, biotechnology companies and secondary screening facilities working to better understand and predict the mechanism of cellular cytotoxicity.