LabAutomation 2006 - SLAS

MP91
David Ferrick
Seahorse Bioscience
Billerica Massachusetts
david @seahorsebio.com
LabAutomation2006
Co-Author(s)
Mark Rothenberg
Min Wu
Chris Braun
Seahorse Bioscience
Extracellular Flux Enables Real-time, Non-invasive Measurements of Cellular Metabolism
Metabolic changes in cells are sensitive and early indicators of biological processes such as proliferation, differentiation, activation, and
apoptosis. We have developed a non-invasive, time resolved, multi-analyte assay for profiling energy utilization in vitro. The Seahorse
Bioscience XF24 instrument simultaneously measures cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)
of cells in microplates in minutes.
Extracellular Flux (XF) assays do not disturb cells and can therefore be repeated frequently to acquire time resolved data, or to compare
basal and drug-stimulated metabolic rates. Measuring the extracellular flux of OCR and ECAR is the basis for calculating ATP turnover,
metabolic rate and the relative contributions of aerobic and anaerobic respiration to total energy production.
Using the Seahorse instrument to profile various cancer cell lines we have demonstrated their well-established increased dependency
on glycolysis. In some cases we observed an apparent defect in aerobic respiration which is novel and consistent with their glycolytic
phenotype.
We have also developed an XF fatty acid oxidation (FAO) assay using palmitate as the fuel source in C2C12 myocytes. Within minutes
upon the addition of palmitate the ratio of OCR/ECAR is increased. Subsequent addition of the CPT-1 inhibitor, Etomoxir, results in a
minimum 50% decrease of FAO.
Because XF assays are rapid and non-invasive, time-resolved drug response profiles can be easily measured. We are exploring
compounds with several partners in various indications such as cancer and metabolic disease as well as toxicity studies.
MP92
Yu Suen
Beckman Coulter
Fullerton, California
ysuen@beckman.com
Co-Author(s)
Keith Roby, Beckman Coulter Inc.
Konstantin Tsinman, pION Inc.
Automation of Modified Shake Flask Solubility Assay on the Biomek FX
ADMETox Assay Workstation
Early drug discovery in vitro ADME assays can help in rejecting or flagging test compounds that lack good pharmaceutical profiles. The
automated uSOL EVOLUTION96 Assay for solubility and the Double-SinkTM PAMPA Evolution96 Permeability Assay for permeability are
critical ADME profiling assays, and methods for these assays have been developed for Beckman Coulter’s Biomek FX ADMETox Assay
Workstation. This poster describes the Biomek ADMETox Assay Workstation performing the uSOL EVOLUTION96 Assay in 96-well format
with on-deck filtration and integrated SpectraMax* microplate spectrophotometer. The uSOL assay is a novel high-throughput “Modified
Shake Flask” method utilizing UV technology. The patented “self-calibration” protocol eliminates the need for serial dilutions dramatically
increasing throughput and enabling high quality in vitro solubility measurements to be performed early in the drug discovery process. The
instrument allows both kinetic and thermodynamic solubility measurements to be done at different pH values. Kinetic solubility involves
detection immediately after the sample is added to solution, whereas thermodynamic solubility requires longer incubation to reach
equilibrium before detection. The batch setup allows thermodynamic solubility, which provides more “downstream” value, to be measured
without sacrificing throughput of compounds. These automated uSOL and PAMPA assays can be used for high-throughput in vitro
absorption screening in early drug discovery.
The information presented here will include:
• Description of the ADMETox Assay Workstation
• Description of the uSOL automated methods
• Results obtained when using the methods on a standard set of drug compounds.
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