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Automated ApoTox-Glo Assay to Simultaneously Assess Cell Viability, Cytotoxicity and ApoptosisDownload
April 29, 2009
Authors: Sarah Shultz, Andrew Niles, Promega Corporation; Jason Greene, Gaby Neumann, Peter Banks, BioTek Instruments
The ApoTox-Glo assay is comprised of the Promega MultiTox-Fluor™ and Caspase-Glo® 3/7 Assays. The MultiTox-Fluor™ Assay is a non-lytic chemistry that allows measurement of live and dead cells in a single sample well. Specifically, for live cell assessment, live-cell protease activity is measured by the fluorogenic, cell-permeant peptide substrate Gly-Phe-7-amino-4-trifluoromethyl coumarin (GF-AFC). This live-cell protease activity marker labels only live cells because it becomes inactive upon loss of membrane integrity and leakage into the surrounding culture medium, and thus does not contribute to the dead cell measurement. For dead cell assessment, a second protease activity marker, the cell-impermeant peptide substrate bis-(Ala-Ala-Phe)-rhodamine 110 (bis-AAF-R110), is used to measure the activity of a dead-cell protease from cells that have lost membrane integrity and leaked the biomarker into the surrounding culture medium. The Caspase-Glo® 3/7 Assay is a luminescent assay that measures caspase-3 and -7 activities in cultures of cells, which are indicative of apoptosis. The assay provides a proluminescent caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD. This substrate is cleaved to release aminoluciferin, a substrate of luciferase used in the production of light. The amount of light produced correlates with caspase-3/7 activity. Together, these assays provide a researcher with three pieces of data per well (cell viability, cytotoxicity, and caspase activity) which can be used to more accurately profile compound effects on cells.
Synergy™ 2 Multi-Detection Microplate Reader.