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ASSAY TUTORIAL Drug Discovery remains an essential task for early-stage drug discovery. Promega's (www.promega.com) Mitochondrial ToxGlo™ Assay, when com- bined with such galactose cell culture condi- tions provides such a solution. The Mitochondrial ToxGlo Assay is a cell-based assay method that uses sequential- addition, multiplexed assay chemistry for predicting potential mitochondrial dysfunc- tion as a result of xenobiotic exposure (Fig- ure 2). The assay is based on the differential measurement of biomarkers associated with changes in cell membrane integrity and cel- lular ATP levels relative to vehicle-treated control cells during short exposure periods. Cell membrane integrity is first assessed by measuring the presence or absence of a Michele Arduengo (michele.arduengo@ promega.com) is a scientific communications specialist IV at Promega. Web: www.promega.com. Figure 1. Mitochondrial responsiveness to a model mitochondrial toxin in the presence of galactose or glucose: Some cells treated in the presence of glucose may preferentially rely on glycolysis to meet bioenergetic needs and are therefore relatively unresponsive to mitochondrial toxins (Glucose ATP). Cells treated in the presence of galactose must use oxidative phosphorylation to generate ATP and are therefore more responsive to mitochondrial perturbation (Galactose ATP). Oligomycin treatment did not cause changes in membrane integrity in either formulation of medium (Galactose Cytotoxicity and Glucose Cytotoxicity). Shown are data from K562 cells at 10,000 cells/well in a white Costar® 96-well plate. The cells were exposed to oligomycin for two hours. A B distinct protease activity associated with ne- crosis using a fluorogenic peptide substrate (bis-AAF-R110) to measure "dead-cell pro- tease activity." The bis-AAF-R110 Substrate cannot cross the intact membrane of live cells and therefore gives no signal with viable cells. Next, ATP is measured by adding an ATP detection reagent, resulting in cell lysis and generation of a luminescent signal propor- tional to the amount of ATP present. The two sets of data can be combined to produce profiles representative of mitochondrial dys- function or nonmitochondrial-related cyto- toxic mechanisms. Summary Incorporating high-throughput assay chemistry that can detect mitochondrial dys- function early in drug discovery programs provides the opportunity to identify poten- tial mitotoxicants before they reach clinical trials or the market population. Think outside the plate. Billions of samples processed in Array Tape™ C D Imagine what this means for you. Figure 2. Representative profiles of mitochondrial toxicity with the Mitochondrial ToxGlo™ Assay: K562 cells were plated at 10,000 cells/well in white 96-well plates (Costar®) and treated with serial dilutions of compounds resuspended in glucose-free (galactose-supplemented) RPMI 1640 media for two hours. Panel A shows no changes in ATP or membrane integrity (MI), which indicates that the compound is not a mitochondrial toxin. Panel B. The reduction in ATP with commensurate MI changes indicate that the compound is not a mitochondrial toxin; instead primary necrosis is taking place. Panel C. The reduction in ATP with no changes in MI indicates that the compound is a mitochondrial toxin. Panel D. The reduction in ATP with discordant changes in MI indicate that the compound is a mitochondrial toxin. Note: If a decrease in fluorescence, or both fluorescence and luminescence, are observed it is typically due to color quenching interferences adversely affecting assay measures. If the cells are dosed in glucose-containing medium, compounds producing ATP-depletion effects should be counter-screened in galactose-containing medium to rule out inhibition of glycolysis. Volume range: 0.5 - 25.0 uL Array Tape™ is a microplate replacement in the form of a continuous polymer strip, serially embossed with reaction wells in customized volumes and formats including 96- and 384-well arrays. www.DouglasScientific.com Genetic Engineering & Biotechnology News Array Tape applications limited only by imagination. | genengnews.com | October 1, 2012 | 25