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October 2012 Issue 

Featured Core: High-Throughput Screening Core

piwnica_worms_david
David Piwnica-Worms, MD, PhD

Kopan
Raphael Kopan, PhD

The purpose of Siteman Cancer Center’s High-Throughput Screening Core (HTC) is to provide instrumentation and support for high-throughput screening; provide expertise in assay design and implementation (both cell-based and chemistry-based); promote interaction among student and faculty members from various disciplines through user group meetings; and provide training opportunities.

The core was established in 2005 through the collaborative efforts of the Mallinckrodt Institute of Radiology, Department of Cell Biology and Physiology, and Department of Developmental Biology. The core director is David Piwnica-Worms, MD, PhD. Raphael Kopan, PhD, is co-director.

High-throughput screening is the process of testing a system or assay designed by a scientist against a bank or library of chemicals or drugs – or against a set of gene-altering chemicals such as siRNAs – to elucidate more details about that system. The assay may be a cellular biochemical pathway with a specific reporter denoting a change in activity; a change in cell growth, death, shape or mobility; a change in enzyme activity or pathogen infectivity; or any change that can be measured or counted.

In the core, a Beckman-Coulter core automation system with an Orca robot (traveling on a 3-meter rail with a Biomek FX liquid handler, plate washer, dispensers and random-access carousels) provides uniform automated processing of plates. Other instrumentation includes stationary robots, barcode printers and readers, stackers, dispensers and nanoliter liquid handlers, which are all capable of handling 96- and 384-well microwell plates containing cells or chemicals. Six-plate readers are capable of reading high-sensitivity luminescence, fluorescence, TRF, FRET, BRET, FP, absorbance, Delfia and Alpha-Screen in end-point or kinetic modes. Biosafety hoods and incubators are available for cell-based assays, and the clean-room atmosphere and inline robotic CO2-humidified incubator enable critical maintenance during assays, such as drug screens or siRNA gene knockdown.

Any study that benefits from automation is supported, whether it’s cell-based, biochemical kinetic or enzymatic small-molecule effects, or for model organisms such as zebrafish. Available libraries include six siRNA, both human and mouse, including the whole human genome, as well as seven small-molecule and drug libraries, totaling about 150,000 compounds and 80,000 siRNAs.

Two high-content fluorescent scanning microscopes – a GE IN Cell 1000 and a Molecular Devices ImageXpress Micro, each with corresponding powerful analysis software – provide phenotypic analyses of live or fixed cells in almost any format. CCD cameras, laser autofocus and motorized stages enable the capture and analysis of thousands of images an hour in any fluorescent color or in bright field or phase contrast. Powerful computer algorithms analyze images for cellular or subcellular changes defined by the researcher using one of 20 preprogrammed or custom-designed sets of parameters that report data as a tabulated Excel file. Temperature and CO2 control allows time-course imaging in live cells.

The core has provided extensive assay support to a wide variety of research and scientific fields, including cancer, Alzheimer’s disease, host-pathogen interaction, new probes for fluorescent cell imaging, pregnancy outcome prediction, bacterial multidrug resistance and nanotechnology research. More than 50 siRNA assays and 20 small-molecule assays have so far resulted in 14 publications and 12 dissertations. Several other scientists are in the process of follow-up hit validation.

Quality assay design and development, which includes running pilot studies to optimize reliability and statistical relevance, are the keys to successful screening. They are made possible in the core through the availability of highly efficient and accurate automation equipment and by support personnel with extensive experience in mammalian cell protein expression, assay development and automation in both industrial and academic settings.

For more information, contact lab manager Jayne Marasa at marasaj@mir.wustl.edu or 314-362-9373.