MT Cell Viability Substrate Viable cell Reduction X No Reduction NanoLuc® luciferase NanoLuc® Substrate Light Light NanoLuc® luciferase No Light Light Figure 1. RealTime-Glo™ MT Cell Viability Assay overview. NanoLuc® Luciferase and a cell-permeant prosubstrate, the MT Cell Viability Substrate, are added to cells in culture. The MT Cell Viability Substrate is reduced to a NanoLuc® substrate by metabolically active cells. The NanoLuc® substrate diffuses from cells into the surrounding culture medium and is rapidly used by NanoLuc® enzyme to produce a luminescent signal. The signal correlates with the number of viable cells. Dead cells do not reduce the substrate and produce no signal. A. 100,000 150,000 200,000 250,000 300,000 50,000 0 0 20 40 Time (hours) 12h EC50 Value (nM) 3.07 24h 2.29 Incubation Time 36h 48h 2.27 1.99 60h 1.78 72h 1.62 60 80 0.0 –1 0 1 Bortezomib (log nM) Data from specific time points plotted to calculate EC50 at these time points. Figure 2. One RealTime-Glo™ Assay plate yields the same aata as many end-point assay plates. Five hundred A549 cells/well were plated (384-well plate) in 40µl of cell culture medium containing 2X RealTime-Glo™ Reagent. An equal volume of 2X bortezomib was added to achieve the indicated concentrations. Luminescence was monitored for 72 hours. Panel A. Cell viability was monitored every hour on a Tecan Infinite® P200 multimode reader with gas control module 37°C/5% CO2 GraphPad Prism® software, version 5.03. Panel B. Change in cell viability of bortezomib-treated cells compared to the vehicle control at each concentration is shown. ). EC50 values (see Table below Panel A) were determined using Pour commander En savoir plus enews | Juillet 2015 2 3 300nM 150nM 75nM 37.5nM 18.75nM 9.38nM 4.69nM 2.34nM 1.17nM 0.59nM 0.29nM 0nM No treatment control B. 1.5 12h 48h 1.0 24h 60h 36h 72h MT Cell Viability Substrate Dead cell 0.5 Luminescence (RLU) Change in Cell Viability Compared to Vehicle Control (fold) 12420TA 12466MC Pagina 2

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