Research Resources
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Shared Resource Facilities (SRF)High-Content Screening Shared Resource Facility (HCS-SRF)Location: Annenberg 18-21 and Annenberg 18-241 The HCS-SRF provides access to high-content screening techniques based on fluorescent microscopy to the Mount Sinai community. Imaging-based assays, once restricted to cell and developmental biology, have become a critical component in a broad spectrum of basic and clinical research. The development of novel fluorescent probes combined with improvements in robotics and computer-assisted feature recognition and informatics, has facilitated the development of commercial microscopes capable of high-throughput evaluation of image-based assays ("High-Content Analysis"). Automated assays based on microscopy data permit the detection of molecular events in the context of their cellular environment and complement other high-throughput methods based on spectroscopy or flow cytometry techniques that examine, primarily, changes that occur en mass. In contrast to these techniques, assays based on images of intact cells permit the detection of small, localized changes in protein expression or distribution that would be otherwise obscured in the context of pooled cell populations or cell lysates. For instance, using fluorophore-conjugated reporters that alter their subcellular distribution in response to physiological signals, it is possible to quantify changes in cell physiology on a single-cell basis. In combination with automated microwell-based microscopes, these reporters permit the evaluation of libraries of compounds or siRNA reagents on a high-throughput basis using changes in cell-structure as the primary method of evaluation. In addition to serving as the basis for library screens, High-Content Analysis also has applications in the characterization of heterogeneous cell populations, serving essentially as an image-based equivalent of a flow cytometer. Cells can be evaluated not only for the coincidence of the expression of molecular markers, but also for the subcellular distribution of those markers, permitting an unbiased characterization of a population. As with flow cytometry, these data can be used quantify normal or pathological changes that occur within a cell population over time. However, whereas flow cytometry can detect changes in the expression of a molecular marker, High-Content Analysis can measure with far greater sensitivity the translocation of that marker from one compartment to another (eg: cytoplasm to membrane) or the alteration of the codistribution of multiple markers within the cell in the absence of any changes in overall expression. This SRF therefore complements existing high throughput methods at Mount Sinai. Staff will assist end users in assay design, equipment automation, programming and automated feature recognition. We estimate that we will be able to evaluate a library of 14,000-20,000 siRNA (small interfering RNA) pools or small compounds over the course of 3-5 days. siRNA libraries are directed primarily at identifying the critical components of pathways under consideration (drug targets). Compound libraries will be screened to identify drug candidates that modulate those targets. At full capacity, we expect to complete 10-14 full screens per year, including assay design and optimization, primary screens and reevaluation of positive samples. Major instrumentsA new Molecular Devices ImageXpress Ultra Confocal Imaging System, the central resource of the SRF, is equipped with four solid-state lasers (405nm, 488nm, 561nm and 635nm) capable of exciting a wide variety of commonly-used fluorophores in parallel. This system, in combination with feature-recognition software (MetaXpress; Molecular Devices) and informatics software (AcuityXpress; Molecular Devices) permits the evaluation, in a 96-or 384-well format, of libraries of compounds or siRNA reagents. Primary image data are archived on an independent server equipped with four terabytes of local storage. Data are subsequently analyzed on one of three independent analysis workstations. The server and analysis workstations are connected to the primary data-collection system by a gigabit LAN connection to allow real time archiving and analysis of data as they are collected. Additionally, the facility offers access to a Perkin Elmer Fusion ratiometric plate fluorometer/luminometer that permits high-throughput assays using spectroscopic reporters, including FRET-based probes. Finally, automated pipetting is carried out using a Perkin Elmer JANUS 8-channel liquid handler and a Thermo Combi liquid dispenser. System integration is handled by a Thermo/Fisher CataLyst Express plate mover under the control of Polara scheduling software (Thermo/Fisher). Together, this system is able to carry out automated pipetting/experimental setup, data collection and analysis for multiple experiments in parallel. Additional InformationDevelopmental and Regenerative Biology - High-Content Screening ContactsDan Felsenfeld, Ph.D., Co-director Marek Mlodzik, Ph.D., Co-director Shared Resource Facilities |