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U.S. National Institutes of Health
Last Updated: 10/28/10

University of Pittsburgh Specialized Application Center

HTS & HCS Facility: The UPDDI HTS/HCS facility occupies 2,400 ft2 of laboratory space on the 9th floor of the recently constructed Biomedical Science Tower-3 (BST-3). Included in this research space are: the main HTS facility room (1,200 ft2); a dedicated HCS laboratory (412 ft2); a dedicated assay development laboratory (414 ft2); 2 fully functional tissue culture rooms (100 ;ft2 each); one room (201 ft2) for HTS reagent preparation, to house the liquid nitrogen cell store, and HTS consumables storage; and 2 offices (120 sq. ft. each).

Compound Storage and Retrieval: To provide secure storage at -20°C and to maintain the integrity of our compound libraries the UPDDI uses a Matrical MatriMinistore™ with 3 VLM modules to provide automated compound storage and retrieval. The system has a capacity for 12,900 384-well plates, ~ 4.95 M compounds.

Zebrafish Facility: The PSAC has access to 11,000 zebrafish tanks in a fully automated facility for use in compound screening and secondary assay development.

Automated Liquid Handlers for Compound Handling and Assay Implementation: (2) Biomek 2000 automated liquid handling systems, (1) Velocity 11 V-prep liquid handling system with 96-well and 384-well transfer heads, (1) Velocity 11 Bravo liquid handling system with 384-well transfer head, (1) Perkin Elmer Multiprobe 8-Tip with a 16 plate deck configuration liquid handling system outfitted with (1) dual 50-plate magazine plate stacker (1) Perkin Elmer Evolution EP3 liquid handling system equipped with both 96-well and 384-well transfer heads and (2) dual 50-plate magazine plate stackers, (1) Perkin Elmer Janus Multiprobe 8-Tip with a 24 ;plate deck configuration liquid handling system outfitted with (1) dual 50-plate magazine plate stacker, (1) Perkin Elmer Janus MDT liquid handling system equipped with both 96-well and 384well transfer heads integrated with an Abgene automated plate sealer and outfitted with (2) dual 50-plate magazine plate stackers, (1) Titertek Zoom dispenser outfitted with (1) dual 50-plate magazine plate stacker, (1) Titertek MAP-C2 plate washer and reagent dispenser outfitted with (1) dual 50-plate magazine plate stacker, (1) Molecular Devices AquaMax DW4 96- and 384-well plate washer and reagent dispenser integrated with the Synchromax ET plate handler, (1) Perkin Elmer FlexDrop IV reagent dispenser with 50-plate stacker capacity, (1) Perkin Elmer Unifilter-96 Harvester, (2) Biotek Elx405 Plate Washers with (2) dual 25-plate magazine plate stackers, (1) Biotek Microflo Bulk Reagent Dispenser with (1) dual 50-plate magazine plate stacker.

The V-prep, Bravo, EP3 and Janus MDT robotic liquid handling platforms are each equipped with 96-well and 384-well transfer heads that are used for replicating compound master plates, preparing compound daughter plates, transferring diluted compounds to assay plates, adding reagents to assays plates, adding controls to assay plates, and serially diluting compounds for IC50/AC50 determinations. The Multiprobes and Biomek 2000 automated liquid handling systems are used to cherry pick active compounds from compound libraries, reformat compound libraries, and to serially dilute compounds for IC50/AC50 determinations. The Zoom, MAP-C2, AquaMax DW4, and Microflo bulk reagent dispensers are utilized for adding buffer to compound plates and reagents or buffers to assay plates. The Zoom, MAP-C2 and Microflo dispensers are also utilized for automated cell seeding into 96- and 384-well plates. The MAP-C2, AquaMax DW4 and Elx405 (Biotek) plate washing platforms are utilized for automating HCS and ELISA assay formats; cell fixation, cell permeabilization, blocking buffer addition, and plate washing.

HTS Detection Instruments: The UPDDI has (3) Molecular Devices SpectraMax M5 plate readers integrated with dual 40-plate magazine StackMax plate stackers, (1) Molecular Devices Flexstation III kinetic microtiter plate reader, (1) Perkin Elmer EnVision multilabel plate reader with AlphaScreen capability with dual 50-plate magazine plate stackers, (1) Perkin Elmer 96-well 2-detector Topcount scintillation and luminescence counter. The M5, Flexstation III and Envision detection platforms are all multimode detection instruments; absorbance, luminescence, fluorescence intensity, fluorescence polarization, time resolved fluorescence, and time resolved fluorescence resonance energy transfer.

HCS Detection Instruments: The UPDDI has four HCS automated imaging platforms; the ArrayScan 3.5 and ArrayScan VTI (AS-VTI) are field based imaging platforms, and the two ImageXpress Ultra (IXU) platforms are confocal imagers. The ArrayScan® VTI houses a Zeiss 200M inverted fluorescence microscope outfitted with selectable 5x/0.25 NA, 10x/0.3NA, 20x/0.4 NA and 40x/0.5 NA Zeiss objectives. Illumination is provided by a full spectrum (300-2000 nM) Hg-halide arc lamp source (EXFO, Quebec, Canada) and fluorescence is detected with a high sensitivity cooled Orca CCD Camera (Photometrics Quantix). The ArrayScan VTI uses an image-based auto-focus system, and using a fast excitation filter wheel combined with a multi-band emission filter it can image up to six fluorescence channels excited and acquired sequentially. The IXU platforms are fully-integrated point-scanning confocal imaging systems with four solid-state lasers providing up to 4 simultaneous excitation wavelengths (405, 488, 532 or 561, 635 nm) each with a dedicated photomultiplier tube for detection. Images in each fluorescent channel may be acquired sequentially, or with the correct combination of fluorescent probes and filters may be acquired in parallel. The IXU utilizes a dedicated high-speed laser auto-focus system, has a 4-position automated objective changer with air objectives (10x, 20x, 40x & 60x), and the detection pinhole diameter of the confocal optics is configurable in the software.

Robotic Plate Handlers: (1) Zymarker Twister I plate handler, (1) Molecular Devices Synchromax ET plate handler, (1) Caliper Twister II plate handler, all operated with iLink control software. (1) CRS Catalyst Express plate-loading robot operated by Polaris control software.

Servers, Network and Data Backup Systems: The UPDDI network has 9 servers: (2) Dell PE 1850’s Domain controller servers; (1) Dell PE 2850 Windows file server with 8 GB memory and two Dual Core 2.8GHz/2MBx2 CPUs; (1) Dell PE 2850 Oracle Database and IDBS ActivityBase® LIMS server with 8 GB memory and two Dual Core 2.8GHz/2MBx2 CPUs; (1) Dell PE2950 server with 4GB memory and two Dual-Core 2.0 GHz/1333 MHz FSB/4MB Cache CPUs attached to a 6TB SATA disk storage to house the a ArrayScan IV Cellomics Store™ database; (1) Dell PE2950 server with 16 GB memory and two Quad Core 66GHz/1333MHz FSB/4MBx2 Cache CPUs with 16TB SAN storage to house the ArrayScan VTI Cellomics Store database; (1) high-performance Dell 2950 application server with four analysis engines and two high-speed quad-core CPUs to house the Definiens image intelligence suite; (2) Dell PE2850 server with 4 GB memory and two Dual Core 2.8GHz/2MBx2 CPUs to house one PubChem raw data FTP/WEB server to provide the public access to the raw data and images from MLSCN screening campaigns completed by PMLSC. A scalable EMC CX300 Storage Area Network (SAN) solution has been deployed and upgraded to be the central data warehouse with 50 TB of formatted storage configured with 7 TB of high performance fiber channel disk storage, and 43TB of SATA disk storage. The SAN is connected to a PowerVault™ PV-124T tape drive with a 16 slot tape library for data backup and recovery.

The UPDDI network provides a secure environment for capturing, analyzing, storing, and sharing the large volume of data and information acquired during an HTS/HCS campaign. The data and information backup to tape provides a secure source and location for data recovery should it be necessary.

LIMS and HTS/HCS Analysis Software: The UPDDI has (3) IDBS ActivityBase LIMS Biology seats, (3) IDBS XE Seats (3) IDBS XLfit seats, (2) IDBS ActivityBase Chemistry seats, (1) IDBS Protocol Transfer Assistant, (7) IDBS SARview seats, (10) Spotfire® data visualization licences, (3) Leadscope Enterprise 2.4. 15-6 Chemical Structure analysis software seats. Cellomics platinum package of Bio-Application image analysis algorithms (V2 & V3) for their ArrayScan® imaging platforms; Cell Cycle, Cell Health Profiling, Cell Motility, Cell Spreading, Compartmental Analysis AS, Cyto-Cell Membrane, Cyto-Nuc Translocation, Extended Neurite OG, GPCR Signaling, Micronucleus, Molecular Translocation, Morphology Explorer, MP Cytotoxicity, Neuronal Profiling, Neurite Outgrowth, Spot Detector, Target Activation, and Tube Formation. (3) vHCS Discovery Toolbox and (3) vHCS View licenses. Molecular Devices ImageXpress application and informatics software package with (10) MetaXpress and AcuityXpress licenses and the parallel computation Cephalopod package. The ImageXpress Ultra imaging platform also has a portfolio of image analysis modules; Cell cycle, Angiogenesis Tube formation, Granularity, Mitotic Index, Multi-wavelength Cell Scoring, Multi-wavelength Translocation, Neurite Outgrowth, Nuclear Translocation, Transfluor, Translocation, and Translocation enhanced. Definiens image intelligence suite for custom image analysis includes; (1) developer, (1) architect, and (1) Cellenger license with (4) data analysis engines.

The UPDDI has deployed the industry standard ActivityBase 7.0 LIMS from IDBS to register compound libraries, manage the compound inventory, and process and upload the HTS compound and bioassay data from detection instrument platforms into the UPDDI Oracle database. The UPDDI has added three XE modules to the existing clients to support and accelerate HTS/HCS data analysis, permit 1536-well and other template building, and data processing. The Vitesse software and inventory module, which controls the Matrical Ministore automated compound storage and retrieval system has been integrated with ActivityBase. XLfit is used to fit curves for concentration dependence (IC50/EC50) assays. SARview 6 is used to query the database and generate HTS data reports containing the required chemical and biological information. The Spotfire DecisionSite 9.1 package provides the project team with data visualization tools for quality control and data analysis. The Leadscope Enterprise 2.4.6-1 software provides structure-based classification and clustering of the active compounds identified in primary screens and/or follow up assays based on recursive partitioning. Co-investigator Dr. Tong Ying Shun has developed two custom software packages, APAC Reporter and Cytominer, to generate specific UPDDI data reports.