Science Catalog 2012 Life Cell Signaling ApoTox-Glo™ Triplex Assay Product ApoTox-Glo™ Triplex Assay For Laboratory Use. Description: The ApoTox-Glo™ Triplex Assay combines three chemistries to easily assess viability, cytotoxicity and apoptosis events in the same assay well. Viability and cytotoxicity are determined by measuring two differential protease biomarkers simultaneously with the addition of a single nonlytic reagent containing two peptide substrates. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant peptide substrate (GF-AFC Substrate). The substrate enters intact cells, where it is cleaved to generate a fluorescent signal proportional to the number of living cells. This live-cell protease activity marker becomes inactive upon loss of membrane integrity and leakage into the culture medium. A second, cellimpermeant, fluorogenic peptide substrate (bis-AAF-R110 Substrate) is used simultaneously to measure dead-cell protease activity that has been released from cells that have lost membrane integrity. This results in ratiometric, inversely correlated measures of cell viability and cytotoxicity. The ratio of viable cells to dead cells is independent of cell number and, therefore, can be used to normalize data. A second reagent containing luminogenic DEVD-peptide substrate for caspase-3/7 and Ultra-Glo™ Recombinant Thermostable Luciferase is added. Caspase-3/7 cleavage of the substrate releases luciferin, which is a substrate for luciferase and generates light. The light output, measured with a luminometer, correlates with caspase-3/7 activation as a key indicator of apoptosis. Features: • Measure Viability, Cytotoxicity and Apoptosis in the Same Sample Well: Determine mechanism of cell death for cells in the same sample well. • Easily Implement: Assay follows a simple sequential “add-mix-measure” format. • Normalize Data with a Built-In Control: The ratio of the number of live cells/number of dead cells is independent of cell number and normalizes data. This normalization makes results more comparable well-to-well, plate-to-plate and day-to-day. • Flexible and Easily Automated: The volumes of each assay component can be scaled to meet throughput needs and is amenable to automation in 96- and 384-well plates. • Improves Efficiency and Saves on Lab Budget: Reduces cell culture and labor costs by performing three assays in a single well. Storage Conditions: Store all components at –20°C protected from light. Protocol ApoTox-Glo™ Triplex Assay Technical Manual Part# TM322 Size Cat.# Price (Fr) 10 ml G6320 920.00 5 × 10 ml G6321 3220.00 ApoLive-Glo™ Multiplex Assay Product ApoLive-Glo™ Multiplex Assay For Laboratory Use. Description: The ApoLive-Glo™ Multiplex Assay measures both the number of viable cells as a marker of cytotoxicity and caspase activation as a marker of apoptosis within a single assay well to determine the mechanism of cell death. The first part of the assay measures the activity of a protease marker of cell viability. The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (glycylphenylalanyl-amino fluorocoumarin; GF-AFC). The substrate enters intact cells, where it is cleaved by the live-cell protease activity to generate a fluorescent signal proportional to the number of living cells. This live-cell protease becomes inactive upon loss of cell membrane integrity and leakage into the surrounding culture medium. The second part of the assay uses the Caspase-Glo® Assay technology to detect caspase activation, a key biomarker of apoptosis. The Caspase-Glo® Assay provides a luminogenic caspase-3/7 substrate, which contains the tetrapeptide sequence DEVD, in a reagent optimized for caspase activity, luciferase activity and cell lysis. Adding the Caspase-Glo® 3/7 Reagent in an ‘add-mix-measure’ format results in cell lysis, followed by caspase cleavage of the substrate and generation of a ‘glow-type’ luminescent signal produced by luciferase. Luminescence is proportional to the amount of caspase activity present. Features: • Measure Viability and Apoptosis in the Same Well: Accurately determine the mechanism of cell death in less time with less sample. • Easy to Implement: The assay uses a simple ‘add-mix-measure’ format. • Normalize Caspase Data with Viability Control: The ratio of caspase activity to viable cells is useful for determining the extent of caspase activation and for normalizing cell numbers. • Flexible and Easily Automated: The volumes of each assay component can be scaled to meet throughput needs, and the assay is amenable to automation in 96- and 384-well plates. • Reveal cell death even if the window of caspase activity is missed. • Multiplex with Other Assays: The nonlytic nature of the first step of the assay allows further multiplexing with spectrally distinct fluorescent assay chemistries. Storage Conditions: Store all components at –20°C protected from light. Protocol ApoLive-Glo™ Multiplex Assay Technical Manual Part# TM325 Size Cat.# Price (Fr) 10 ml G6410 788.00 5 × 10 ml G6411 2758.00 26 For complete and up-to-date product information visit: www.promega.com/catalog Pagina 29
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