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This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.
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The Mitochondrial ToxGlo™ Assay is a cell-based assay method that employs a sequential addition, multiplexed assay chemistry for predicting potential mitochondrial dysfunction as a result of xenobiotic exposure. The assay is based on the differential measurement of biomarkers associated with changes in cell membrane integrity and cellular 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 distinct protease activity associated with necrosis using a fluorogenic peptide substrate (bis-AAF-R110) to measure "dead cell protease 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 proportional to the amount of ATP present. The two sets of data can be combined to produce profiles representative of mitochondrial dysfunction or non-mitochondrial related cytotoxic mechanisms.
Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) methods. To achieve optimal mitochondrial responsiveness, it may be necessary to refine cell culture conditions. Replacing glucose-supplemented medium with galactose-containing medium may increase cellular oxygen consumption and augment mitochondrial susceptibility to mitotoxicants.
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 2 hours.
Mitochondrial ToxGlo™ Assay produces profiles that are consistent with mitochondrial toxicity and discernible from other non-mitotoxic mechanisms of cell death. The assay is easy to implement as it uses a simple sequential “add-mix-read” format, and assays are performed directly in cell culture plates using standard multimode detection instrumentation.
A flexible and easily automated assay, the volume of reagent addition can be scaled to meet throughput needs; the assay is amenable to automation in 96- and 384-well plates.
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 2 hours. Panel A. The changes in ATP or membrane integrity (MI) are detected, 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.
"Add-mix-measure" protocol is fast and easy to implement. No parallel processing in glucose-containing medium.
Mitochondrial ToxGlo™ Assay Technical Manual
PDF (432 KB)
ATP Detection Buffer
ATP Detection Substrate
PDF (0 B)
Search for Specific Certificate:
U.S. Pat. Nos. 7,083,911, 7,452,663 and 7,732,128 and other patents.
U.S. Pat. Nos. 6,602,677, 7,241,584, 8,030,017 and 8,822,170 and other patents and patents pending.
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