Science Catalog 2012 Life Cell Signaling Mitochondrial Function Assay Mitochondrial Toxicity Assay Product Mitochondrial ToxGlo™ Assay For Research Use Only. Not for Use in Diagnostic Procedures. Description: The Mitochondrial ToxGlo™ Assay is a cell-based assay method that employs 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 abscence 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 nonmitochondrial (glycolysis) methods. To achieve optimal mitochondrial responsiveness, replacing glucose-supplemented medium with galactosecontaining medium may increase cellular oxygen consumption and augment mitochondrial susceptibility to mitotoxicants. A. 100 150 50 0 –7 C. –6 –5 Log [imipramine], M 100 150 50 0 –7 Log [CCCP], M –6 –5 D. 100 200 300 400 500 600 0 –7 Log [antimycin], g/L –6 –5 –4 Cytotoxicity ATP B. 100 150 200 250 50 0 Size Cat.# Price (Fr) 10 ml G8000 Pls. Enq. 100 ml G8001 Pls. Enq. Features: • Distinguish Primary Mitochondrial Dysfunction from Secondary Cytotoxic Events: Cell-based, multiplexed method measures ATP in conjunction with a membrane integrity biomarker to distinguish primary mitochondrial dysfunction from secondary cytotoxic events. • Predictive for Mitochondrial Toxicities: Produces profiles that are consistent with mitochondrial toxicity and discernible from other nonmitotoxic mechanisms of cell death. • Easy to Implement: The assay uses a simple sequential “add-mix-read” format to assess toxicity in the sample well. • Fast: Quickly assess potential mitochondrial liabilities in under an hour. • Cost-Effective: Assays are performed directly in cell culture plates using standard multimode detection instrumentation. • Flexible and Easily Automated: The volume of reagent addition can be scaled to meet throughput needs; the assay is amenable to automation in 96- and 384-well plates. Storage Conditions: Store the Mitochondrial Tox-Glo™ Assay components at –20°C. Protocol Mitochondrial ToxGlo™ Assay Technical Manual Part# TM357 –6 –5 Log [digitonin], g/L –4 Representative profiles of mitochondrial toxicity with the Mitochondrial ToxGlo™ Assay. K562 cells were plated at 10,000 cells/well in 96-well plates and treated with serial dilutions of compounds resuspended in glucose-free (galactose-supplemented) RPMI 1640 medium for 2 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. 46 For complete and up-to-date product information visit: www.promega.com/catalog Percent Vehicle Control Percent Vehicle Control Percent Vehicle Control Percent Vehicle Control 9955MA Pagina 49
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