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High Content Analysis - Day 2


Thursday, January 13

7:30-8:15 Breakfast Presentation
From Differentiation to Death: Using Automated Microscopy to Explore Cell PhysiologySponsored by
American Pharmaceutical Review

David W. Andrews, Professor of Biochemistry and Biomedical Sciences & Canada Research Chair in Membrane Biogenesis, McMaster University

The application of advanced imaging techniques to high-content analysis permits increasingly sophisticated questions to be addressed via large-scale biological screens. Using automated high-throughput microscopes the effect of siRNAs, shRNAs, small molecules, or genetic knockout libraries can be rapidly ascertained. The accessibility of these techniques has spurred major developments in the fields of systems biology, functional genomics and chemical genetics. I will describe our use of high-content imaging to quantify complex biological processes including: the differentiation of human stem cells, radiation induced chromatin damage, autophagy, cellular stress responses, apoptosis and for determining the molecular mechanism(s) of drugs.

8:30-9:30 ThinkTank Roundtable Discussions

Roundtable discussions, led by expert facilitators, are open to all delegates.

Table 1: Compound Screening

Moderator: Joseph Zock, Thermo Fisher Scientific

Table 2: Data Analysis and Management

Moderator: Karol Kozak, Ph.D., ETH Zurich

Table 3: Live-Cell Imaging

Moderator: David W. Andrews, Ph.D., McMaster University

Table 4: New Biological Models

Moderator: Paul Sammak, Ph.D., University of Pittsburgh

Table 5: Flow Cytometry

Moderator: J. Paul Robinson, Ph.D., Purdue University

Table 6: Novel Probes and Biosensors

Moderator: Paul A. Johnston, Ph.D., University of Pittsburgh

Table 7: Pathway Analysis

Moderator: Vivek C. Abraham, Ph.D., Abbott Laboratories

Table 8: Toxicity Profiling

Moderator: Peter J. O'Brien, D.V.M., Ph.D., University College, Dublin

Table 9: Neuronal Screening

Moderator: John L. Bixby, Ph.D., University of Miami

9:30-10:00 Reports from ThinkTank Roundtable Discussions

10:00-10:30 Award Presentations

10:30-11:45 Coffee Break with Exhibit and Poster Viewing

Flow Cytometry

Chairperson's Opening Remarks

J. Paul Robinson, Ph.D., SVM Professor, Cytomics, Biomedical Engineering; Director, Purdue University Cytometry Laboratories, Purdue University

11:45-12:10 pm High-Content/High-Throughput Drug Screening: Complete Analysis of a 384 Well Complex Multicolor Flow Cytometry Assay in Less than a Minute

J. Paul Robinson, Ph.D., SVM Professor, Cytomics, Biomedical Engineering; Director, Purdue University Cytometry Laboratories, Purdue University

An automated high-content screen is one that requires a great deal of optimization and standardization of many procedures. Successful creation of the assay and collection of the data is just the beginning of a good HC screen. Possibly the most difficult part is analyzing the data and reducing the very large data set quickly into something meaningful. When evaluating flow cytometry data, most current procedures operate in a purely vertical fashion. This cannot be translated into very large volume processing, since all of the concepts are applied at the local, rather than global levels. Creation of an analytical process capable of handling millions of cells with multiple-variable data sets requires a fully parallel analysis concept. This means creating analytical processes in a representative fashion whereby the solutions are translated as algorithms within a logical sequence applied simultaneously to the entire plate if appropriate. Using this technique, it is possible to facilitate direct visualization of results in a way that is immediately understandable and also very efficient. Spreadsheet output allows translation to alternative data handling systems of virtually any informatics network. Advanced statistical processes are incorporated within the core of this technology allowing rapid “what-if” questions to be posed to test questions of interest. Despite the speed and complexity of this technology, it is still possible to rapidly identify and plot any cell in any well for any parameter or combination of parameters if desired. This presentation will describe the implementation of such a system from the automation of plate setup, to the very high speed flow cytometry to the final analysis. Assays will be demonstrated using multicolor live-cell assays. The automation of this technology allows us to run and completely analyze well over 20,000 multiparameter flow cytometry samples in one day. This opens up the possibility of performing studies that heretofore would have been untenable.

12:10-12:35 High-Throughput Flow Cytometry for Small Molecule Discovery in Complex Cell and Molecular Suspensions

Larry A. Sklar, Ph.D., Director, University of New Mexico Center
for Molecular Discovery

The University of New Mexico Center for Molecule Discovery identifies and implements novel applications of the HyperCyt flow cytometry platform for high-content, high-throughput small molecule discovery. Flow cytometry is recognized for its unique ability to analyze complex target populations in cell and molecular screening where multi-parameter analysis is required. Recent applications include complex cells mixtures for immunological and oncological targets, pathway analysis, protein trafficking, and host-pathogen interactions as well as multiplexed bead-based molecular targets.

12:35-1:05 Flow Cytometry and
High-Content Imaging to Identify Markers
of Macrophage DifferentiationSponsored by
BD Biosciences

Dev Mittar, Ph.D., Senior Scientist, BD Biosciences

The identification and characterization of cell surface CD markers present on different immune cells can provide valuable tools to distinguish cell types for various research and clinical applications as well as for the understanding of immune cell function. We have used the BD Lyoplate™ Human Cell Surface Marker Screening Panel to screen undifferentiated human THP-1 monocytes by high-throughput flow cytometry using BD LSR™ or BD FACSCanto™ II systems with BD high-throughput samplers (HTS), and to screen differentiated THP-1 macrophages using a BD Pathway™ high-content imager. We identified and compared the CD expression profile of these two differentiation states.

HCA for Pathway Analysis

Chairperson's Opening Remarks

Vivek C. Abraham, Ph.D., Group Leader, Lead Discovery, Abbott Laboratories

11:45-12:10 pm Development of a High-Content Screening Assay Panel to Enable Dissection of Mechanism of Action of Compounds that Inhibit Tumor Cell Growth

Vivek C. Abraham, Ph.D., Group Leader, Lead Discovery, Abbott Laboratories

Phenotypic and targeted high-throughput screens designed to discover compounds that inhibit tumor cell growth typically yield large numbers of hits. However, there is an unmet need to be able to rapidly define the mechanism of action of hit compounds. We have developed an HCS assay panel that involves measurement of 10 distinct, biologically interpretable cellular parameters that report on the major mechanisms of cell growth inhibition. These include apoptosis (mitochondrial and non-mitochondrial), cell cycle disruption, DNA damage and multiple cellular morphological changes. This presentation describes the design, development and validation of this assay panel. Novel approaches to a) display the data to ease interpretation and b) cluster compounds based on likely mechanism of cell growth inhibition will also be presented. We expect this general approach to be valuable in drug discovery across multiple therapeutic areas.

12:10-12:35 Image-Based Genome-Wide siRNA Screening to Study Cellular PathwaysSponsored by
BD Biosciences

Marc Chambon, Ph.D., Assay Development Staff Scientist, Biomolecular Screening Facility, Swiss Federal Institute of Technology of Lausanne

Through two high-content genome-wide siRNA screening projects we performed, one dealing with centrosome duplication, the other one concerning cellular lipid distribution, the complexity to develop and validate this kind of cell-based assay will be addressed. Results for the different critical steps will be presented including long-term cell growth in 384-well plates, automated siRNA transfection, complex staining protocols (4 steps) and image acquisition (4 colors, multi z sections), as well as image analysis and data management. Finally, the added value of high-content siRNA screenings will be discussed.

12:35-1:00 Re-Inventing the HIV Cell Fusion Assay Using High Content

Lisa Smith, M.S., Scientist, In Vitro Pharmacology, Merck Research Labs

The HIV cell fusion assay is a surrogate system for detecting viral entry. A T-cell tropic HIV cell fusion assay was established using U2OS cells expressing the envelope glycoprotein gp160 from HIV NL4-3 (transduced by BacMam virus) and HeLa cells expressing CD4 and CXCR4. High-content analysis of the cell fusion event is based upon a Gal4/VP16-activated beta-lactamase signal, using the Pathway855 (BD Biosciences) or the Acumen eX3 (TTP Labtech). Measurement of changes in morphology associated with cell fusion was combined with beta-lactamase activity to show robust assay statistics in 384-well and 1536-well plates.

1:00-2:00 Lunch on Your Own

2:00-6:00 BD Biosciences User Group MeetingSponsored by
BD Biosciences

2:00-6:00 PerkinElmer User Group MeetingSponsored by
American Pharmaceutical Review

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