Revision date: 9/11/2006

The Cell and Media Core provides services related to cell culture, preparation of cell media and solutions, and glassware washing and sterilization. The facility strives to provide services that are efficient, convenient, and cost effective to investigators. The core can provide researchers with large quantities of monolayer and suspension cells, and has facilities for specialized cell culture such as spinner and roller bottle culture, insect cell culture, and EBV transformations. The core also has a mycoplasma and quarantine facility and offers mycoplasma testing on a regular basis.

The media production lab is a convenient and economical service with outstanding quality control. The core can also accommodate custom media orders.

The glasswash facility handles washing and sterilization of all types of glassware for all the labs in the LRI. The facility maintains a stock of all types of sterile laboratory glassware and pipettes in a common storage area. Custom autoclaving needs can be easily accommodated.

Revision date: 7/2/2008

The Lerner Research Institute Computing Services group provides support for the desktop, server, and network-computing infrastructure of the Institute. The combined supported user base is greater than 1,000. Platforms supported include Windows 9x/NT/2000/XP, Windows Server 2000 and 2003, Macintosh OS 7 to 9 and Macintosh OS X, and the Unix variants SGI Irix, Sun Solaris, and Linux. The group provides support for shared network disk space and backup, various Internet communication and collaboration protocols, and application and print servers. It maintains domain name services for the /17 RFC 1918 IP allocation assigned to the Lerner Research Institute by the Cleveland Clinic Information Technology Division and administers the lerner.ad.cchs.net subdomain of Cleveland Clinic's Active Directory forest. The Desktop group provides custom support of the scientific computing workstations across the Macintosh and Windows platforms. The Windows Server team specializes in supporting the Windows Server environment, including domain controller, file, and print, and application services, and Windows and Unix backup. In cooperation with Unix and Desktop support, the Windows Server Team works to provide a collaborative cross-platform computing environment. Unix workstations and servers currently participate in a unified computing structure supported by a dedicated Unix administration team.

Online disk storage exceeds 15 terabytes, which is fully replicated in hardware. A quad-head 670-cartridge LTO-2 tape robot and a quad-head 120-cartridge LTO-2 tape library provide backup services. Shared advanced computing resources include a quad-processor SGI 3400 with 4 gigabytes of memory, a quad-processor SGI Origin 200 with 4 gigabytes of memory, a dual processor SGI Origin 200 with 2 gigabytes of memory, a quad processor Sun 420R with 4 gigabytes of memory, and a 14 node Linux cluster. Other Windows, Linux, SGI, and Solaris server resources are available for special hardware or software needs.

Basic network service to desktop computers is 100 Mbps Ethernet, with 1 Gbps Ethernet available where needed. The Cisco-based network has a redundant 20 Gbps portchannel backbone. Distribution switches link to the backbone via redundant 2 Gbps portchannel connections, or in some cases redundant 1 Gbps connections. Uplinks to the Cleveland Clinic Core are via redundant 2 Gbs portchannel connections. Internet service is provided via the Cleveland Clinic 150 Mbs link.

Revision date: 9/11/2006

The Imaging core provides digital imaging, confocal and fluorescence microscopy, and laser microdissection. The core has three Leica DMR fluorescence microscopes equipped with Q-Imaging Retiga EXi cooled CCD cameras and Image ProPlus Capture and Analysis software for transmitted light (i.e. brightfield, phase contrast, differential interference contrast, darkfield) or fluorescence imaging. Two of the microscopes are designed for capturing images from fixed specimens on slides. The third microscope is inverted and can be used for short-term observation and imaging of live cells in flasks or culture dishes. For live time-lapse imaging over hours to days we have an inverted Leica DMIRB microscope equipped for both transmitted and fluorescence imaging with a heated stage and Leica environmental chamber, computer-controlled Sutter Instruments Lambda-10 motorized optical filter wheel, Roper CoolSnap HQ cooled CCD camera and Universal Imaging's Metamorph software. All microscopes are equipped with a full range of Chroma filters for fluorescent protein imaging.

For confocal microscopy, the Imaging core has two uniquely capable Leica TCS-SP spectral laser scanning confocal microscopes each equipped with four lasers for fluorescence excitation at up to eight separate wavelengths. This allows us to visualize multi-labeled components of a specimen simultaneously to compare the three-dimensional relationships between them. For example we can visualize fluorescently labeled proteins and compare the quantity and localization relative to other labeled cellular components. For post-processing and analysis of confocal data we have several Dell Workstations and a Mac G5 running Improvision's Volocity software. These systems allow for the reconstruction of confocal optical slices into three-dimensional images using a variety of rendering methods. They also allow us to analyze and quantitate, three-dimensionally, the morphology and distribution of various labeled structures.

The core also has a wide range of software, scanners, and printers for optimal image manipulation.

Revision date: 9/11/2006

The electron microscopy section of the Imaging core provides multiple services including embedding, thin and thick sectioning, pre and post-embedding immunoEM, sample observation in our FEI CM12 transmission electron microscope, and photomicrography. The core also maintains and trains users on the JEOL Scanning electron microscope, which is additionally equipped with elemental analysis. Users can get help fixing, dehydrating, critical point drying and sputter coating their samples in preparation for viewing and imaging in the SEM.

Revision date: 9/11/2006

The Electronics Core is a full service electronics design, fabrication and testing facility. It is staffed with two electronics engineers, and two technicians specializing in the development of innovative electronic solutions. Services provided include: circuit and electro-mechanical system design; printed circuit board design and layout; circuit fabrication and testing; custom system control software; NIST traceable calibration of pressure, temperature and voltage; repair and modification of equipment; assistance with experiment set up and data gathering in both the OR and laboratory settings;

The facility resources include: Lindgren RF shielded room; Agilent 4 channel 2GS/s oscilloscope; Protel EDA schematic capture and PCB layout software; T-Tech milling PCB prototyping system; extensive electronic component inventory; surface mount PCB assembly station; integrated calibration system including HP precision LCR meter, HP precision function generator, Agilent arbitrary waveform generator, Mensor 200 psi pressure calibration system, Ectron thermocouple simulator, and voltage calibrator.

The core also offers consultation in the areas of electronics, data collection and laboratory systems.

Revision date: 03/19/2012

The Flow Cytometry Core provides investigators with a resource for analytical and preparative studies of cells using flow cytometry. The Core is equipped with a Becton- Dickinson LSR II capable of 12 color and 14 parameter acquisition with 5 lasers, a Becton- Dickinson LSRFortessa capable of 18 color and 20 parameter acquisition with 5 lasers and High Throughput Sampler (HTS), an Invitrogen Countess™ automated cell counter, and two Becton-Dickinson FACScans capable of 3 color and 5 parameter acquisition with an Argon ion laser. Additionally, a Becton-Dickinson Special Order BD FACSAria II high performance, fixed alignment cuvette cell sorter, consisting of 5 lasers, providing 16 parameters, is used for cell sorting. The BioProtect III Walk In Clean Air and Containment Cabinet houses the FACSAria II and offers personnel, product, and environmental protection from potentially aerosolized biological hazards.

Data is acquired as FCS 3.0 using BD Cell Quest on the FACScans and BD FACSDiva Software on the LSR II, LSRFortessa and Aria II. Data can be imported/exported as FCS 2.0 or 3.0 when using FACSDiva. List mode acquisition and storage of data allows for analysis and presentation many different formats according to the needs of the investigator. Data files are permanently stored in personal folders on the LRI servers, readily available to all LRI employees with proper logon accounts. CellQuest, FlowJo and ModFit software are provided for analysis.

• Flow Cytometry Core Grant Information

Revision date: 9/11/2006

This core provides services and equipment to facilitate gene expression profiling and quantitation. The core uses Affymetrix technology and provides full services, from cRNA preparation to data reduction/data analysis, as well as access to server-based data mining and annotation tools. SNP analysis using Affymetrix arrays is also available, and includes sample preparation and processing as well as assistance with analysis.

The core also provides access to an ABI 7900 instrument for quantitation of gene expression by real-time RT-PCR; full services include brokerage of ABI RT-PCR assay kits and reagents, sample preparation, RT-PCR set-up for relative and absolute quantitation, and help with data interpretation/analysis. SNP analysis using the ABI system is also provided, and includes sample preparation in 384-well format (high throughput enabled by Matrix robotics), endpoint plate reading (high throughput enabled by ABI autoloader), measurement of real time spectral data for initial validation, basic PCR execution, and help with analysis.

The core uses a Nanodrop spectrophotometer and Agilent Bioanalyzer for accurate quantitation and assessment of RNA quality. In addition, the core regularly organizes a 3 credit-hour course on expression profiling, and regular software workshops and training.

Revision date: 9/11/2006

The Genomics Core is a recent addition to the CCF / LRI Core services community and will provide research groups access to state-of-the-art genomics capabilities (sequencing, genotyping and gene expression arrays).

To support high-throughput sequencing and traditional genotyping projects the Genomics Core is equipped with an ABI 3730xl DNA analyzer. To support array-based genotyping projects the Core has installed an Illumina Beadstation which also serves to support gene expression projects. The Illumina system became operational in August 2006. In addition, for custom gene expression projects the Genomics Core has a cDNA Microarray platform that uses sets of human and mouse custom arrays slides whose probe content is aimed at gene expression studies in the areas of cancer and immunology.

Revision date: 9/11/2006

The Hybridoma Core provides a resource for both development and production of monoclonal antibodies, including consultation regarding all aspects of monoclonal antibody production such as epitope design. Services are performed under strict QC guidelines appropriate for antibodies that may have commercial potential. The core uses both stationary (T flasks) and suspension (roller bottle/spinner flasks) cultures for the production of monoclonal antibodies from hybridoma cell lines. Hybridoma cell lines can also be grown in Integra flasks, where the cells are maintained in a compartment separated from the media reservoir by a selective membrane, and the antibody is secreted in a relatively small volume of the extracapillary space. The advantage of this system is that the antibody is 50-100 fold more concentrated and of high purity.

The HCF also offers polyclonal antibody production on a limited basis. The polyclonal antibodies are produced in rabbits, so capacity is determined by the availability of housing in the BRU (Animal Facility).

Revision date: 9/11/2006

The LRI provides and maintains multiple shared instruments that are available to all CCLCM investigators. These include 2 Molecular Dynamics phosphorimagers, a Storm Imager, a Bio-Rad Multi-Imager, several ABI and Bio-Rad cyclers for real-time RT-PCR analysis, a Shepard cesium irradiator, and a Pantak c abinet X-ray irradiator capable of delivering multiple Grays to small experimental systems.

Revision date: 9/11/2006

The Mass Spectrometry Core is designed to meet the growing needs of investigators for development of analytical methods for identification and quantification of biomarkers in plasma, tissue and other biological materials. The main focus of this core is quantitative analysis of small molecules in complex matrices and structural characterization of small compounds. The core offers the service for investigators to develop analytical methods for i dentification of novel bioactive compounds, and for quantification of biomarkers in biological materials like plasma, urine and tissues.

The Core is equipped with a triple quadrupole mass spectrometer (Micromass Quattro Ultima) and two HPLC systems. The mass spectrometer has two ionization sources available: electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). The effective mass range is 4180 Da for singly charged species. The Waters model 2690 HPLC is equipped with a Waters 996 Photodiode Array Detector and a 5 tray, 24 sample/tray auto-sampler. This instrument is coupled with Quattro Ultima mass spectrometer. The Beckman 126 HPLC is equipped with a 96 samples auto-sampler, both a 168 Photodiode Array Detector and a Jasco FP-1520 fluorescence detector.

Revision date: 5/28/2009

The MBT Core offers consultation and services in the areas of customized peptide synthesis, peptide modification, purification and quality analysis, N-terminal protein sequencing by Edman degradation, CD Spectroscopy and biomolecular interaction analysis and equilibrium and kinetic measurements. These services are described in more detail below:

The peptide synthesis services include peptide design, synthesis, cyclization, HPLC purification and various types of modifications of synthetic peptides. Synthesis is carried out at 50 µmole, 100 µmole or 250 µmole scales on Omega 396 and ABI 431A synthesizers. The Core also synthesizes multiple antigen peptides (MAP). Quality is routinely confirmed by HPLC analysis and mass analysis by MALDI-TOF.

N-terminal Edman sequencing is performed on a Procise Model 492 Sequencer. To obtain an unambiguous sequence of an unknown protein about 5 pmol or more of protein / peptide is required. Purity and quantity are determining factors for successful sequencing. For proteins with blocked amino termini, the Core offers chemical or enzymatic digestion and separation of polypeptide fragments of such samples to obtain de novo N-terminal sequence information of the fragments.

The Core offers training and facilitates biomolecular interactions analysis using the Biacore 3000 system, based on the principle of Surface Plasmon Resonance (SPR). The Biacore 3000 is used for measuring the interactions of macromolecules with each other or with small ligands, and for equilibrium and kinetic measurements, competition assays, and epitope mapping. The core provides hands-on training on both Biacore 3000 and the BIAevaluation software to graduate students and fellows.

The Core maintains and provides training to use CD spectroscopy. CD Spectroscopy is a valuable instrument for rapid analysis of structural and conformational changes in a protein upon perturbation by mutation, temperature, pH and buffers. CD spectra arising due to peptide bond transitions in the "far-uv" spectral region (190-250 nm) provide information on secondary structure of protein, while CD spectra of proteins in the "near-uv" spectral region (250-350 nm) provide information on certain aspects of tertiary structure.

Revision date: 9/11/2006

The Polymer Core supports research and medical device development efforts for which polymeric and biologic materials are required. The laboratory uses synthetic and natural polymeric materials such as polyurethane, silicone, PVC, epoxies, polyolefins, and natural rubber. They also specialize in the processing of biologic materials such as gelatin, hyaluronan, and pericardial tissue. After fabricating the materials or devices, the Polymer Laboratory personnel works closely with researchers and clinicians to support the in vivo evaluations of the new technology. The core has participated in projects to manufacture such diverse items as components for artificial heart programs, such as pump diaphragms, inflow and outflow cannulas, heart valves, sewing cuffs, and blood-compatible internal coatings, transparent devices for flow pattern studies, and implantable parts with pore-textured surfaces for optimal cell/tissue growth.

Revision date: 9/11/2006

The Proteomics Core is directed by Dr. Michael Kinter, an expert in the analytical applications of tandem mass spectrometry. Dr. Kinter has directed academic mass spectrometry core laboratories since 1986 and is highly experienced in the operation of multiple mass spectrometry systems including high-resolution magnetic sector instruments, tandem quadrupole instruments, ion trap instruments, and time-of-flight instruments. He has wide-ranging experience with a variety of classes of analytes of interest in biomedical investigations and especially proteins and peptides. The laboratory routinely uses high-sensitivity tandem mass spectrometry methods to sequence and identify protein bands detected in polyacrylamide gels by either Coomassie blue- or silver-staining.

Major equipment in the laboratory includes three capillary column HPLC-electrospray ionization-tandem mass spectrometry systems (a ThermoFinnigan LCQ-deca, a ThermoFinnigan LTQ, and a Micromass Q-ToFmicro), and Maldi-ToF mass spectrometry system (a Micromass TofSpec 2E). All of these instruments are dedicated to high-sensitivity protein sequencing and identification experiments. In addition to these mass spectrometry systems, the laboratory contains a variety of electrophoresis equipment, including systems for isoelectric focusing from both Pharmacia and BioRad, and appropriate SDS-PAGE systems, with scanning equipment, computer facilities, and software (PDQuest) for the analysis of the 2D gels that are produced.

Revision date: 9/11/2006

The Mechanical Prototype Laboratory (MPL) provides a variety of engineering, design, fabrication, customization, and repair services for mechanical devices and equipment for basic research and clinical activities. Our staff is experienced in all phases engineering, design, and fabrication. We are proficient in precision machining of metals and plastics, welding of structural and exotic metals, and mechanical repair.

The laboratory personnel can work from concept sketches to design and fabricate new devices, test fixtures, and equipment. Documentation of the newly created part or device is also available through two- or three-dimensional computer-aided design (CAD) drawings. The software we use is Pro-Engineer, Solid Works, and AutoCAD. These CAD drawings can then be converted into computer aided manufacturing ( CAM ) programs that are directly fed into the Computer Numerical Control (CNC) machines or Rapid Prototyping machines to fabricate the desired parts. These parts can then be inspected on our CNC Coordinate Measurement Machine.

The laboratory features state-of-the-art CNC equipment for the fabrication of highly complex devices or when identical multiple components are required. The CNC equipment list includes: a 5-axis vertical machining center, a 4-axis lathe, and a 4-axis Wire Electrode Discharge Machine. A Coordinate Measurement Machine, which enables precision inspection of devices fabricated, is also located in the laboratory. Machining equipment includes: 2 ½-axis CNC milling centers, a 2-axis CNC lathe, 3-axis manual milling machines, manual lathes, universal tool grinder, and surface and sectioning grinders.

The equipment at our disposal allows us to create high-precision devices for a wide range of clinical or research applications. Examples, recent projects include the engineering, designing, and fabrication of: 1) a treadmill to simulate multi axis movement in a 3-dimensional environment; 2) a device to harvest corneal stem cells; 3) a new style probe used in Deep Brain Stimulation; and 4) a new ablation probe insertion device for the prostate.

Revision date: 9/11/2006

Small Molecule Screening Core (SMSC) provides the researchers at CCF and other biomedical research centers in Cleveland area with the expertise, technology, instrumentation and reagents necessary for the screening of the library of small molecules for biologically active compounds in a variety of readout systems. SMSC owns the diverse historical chemical library of 34,000 compounds manufactured by Chembridge Corporation, world recognized lieder in this field. The core can provide help with the adjustment of readout systems for both cell-based and biochemical assays and establishment of screening conditions based on a pilot small-scale screening. If successful, the established system is used for a full-scale library screening in 96-well format followed by generation of the database with the results. SMSC is equipped with laminar flow hood, CO2 incubators and Multidrop 384 (Thermo/Labsystems) to assist in plating and growing cells in 96-well format. Wallac 1420 VICTOR2 multilabel reader can be used to collect the data in variety of readout systems. Additionally SMSC can provide the information about cytotoxicity of selected hits.

SMSC also offers help with data processing and planning further steps in optimization of isolated chemicals, including primary chemoinformatics analysis, generation of virtual libraries of analogues and consulting on the tactics of compound characterization, including connection with outside partners.