MITOCHONDRIAL TOXICITY Determine the Mechanism of Toxicity The Mitochondrial ToxGlo™ Assay is a cell-based assay that monitors mitochondrial toxicity based on differential measurement of biomarkers associated with changes in cell membrane integrity and cellular ATP levels relative to vehicle-treated controls. The assay is performed in a single-well, first by assessing membrane integrity using a fluorescent substrate then measuring ATP concentration, using a lytic bioluminescent ATP detection reagent. The bioluminescent signal is proportional to the amount of ATP present in living cells, and the fluorescent signal indicates loss of membrane integrity. The two sets of data are combined to produce profiles representative of mitochondrial dysfunction or nonmitochondrialrelated cytotoxic mechanisms. 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 –6 –5 Log [digitonin], g/L –4 Learn More © 2015 Promega Corporation. CellTiter-Glo and NanoLuc are registered trademarks of Promega Corporation. ADP-Glo, CellTiter-Fluor, CellTox, RealTime-Glo and ToxGlo are trademarks of Promega Corporation. alamarBlue is a registered trademark of Life Technologies Corporation. Costar is a registered trademark of Corning, Inc. GraphPad Prism is a registered trademark of GraphPad Software, Inc. GravityPLUS is a trademark of InSphero AG. Infinite is a registered trademark of Tecan AG Corporation. Matrigel is a registered trademark of Discovery Labware, Inc. Products may be covered by pending or issued patents or may have certain limitations. Please visit www.promega.com for more information. Contact us enews | April 2015 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 (galactosesupplemented) RPMI 1640 media 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. 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 counterscreened in galactose-containing medium to rule out inhibition of glycolysis. Percent Vehicle Control Percent Vehicle Control Percent Vehicle Control Percent Vehicle Control 9955MA Pagina 9
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Promega Benelux - Monthly online Magazine - April 2015 edition Lees publicatie 100Home