Grant Descriptions

The Antibody Development laboratory provides center investigators with custom designed monoclonal antibodies. Development activities focus on several areas: a) design of antigens, immunization and screening strategies, b) hybridoma cell line production via traditional methods, c) optimization of existing hybridoma cell lines by selection cloning and d) cloning, expression and engineering of single chain antibody variable fragments (scFvs) or scFvs containing biological reporter molecules such as green fluorescent protein.

The Antibody Development laboratory provides center investigators with custom designed monoclonal antibodies. In most if not all cases, these are necessary reagents that are not commercially available. Development activities focus on several areas:

• Design of antigens, immunization and screening strategies. This includes peptide design and decisions regarding the selection of MAPs versus KLH (or other carrier protein)-conjugates, antigen purification and solubilization, and the optimization of screening protocols.
• Hybridoma cell line production via traditional methods. This includes initial screening of polyclonal antiserum via ELISA, Western blot, and immune precipitation, fusion, post-fusion screening, positive well expansion and subcloning of hybridomas secreting monoclonal antibodies with the desired specificity.
• Optimization of existing hybridoma cell lines by selection cloning.
• Production of monoclonal antibodies in species other than mice.
• Services to wean hybridoma cell lines off selection media and into a variety of basal and serum free media for use in mini-bioreactors (BD Cellline 1000 Flasks), immune precipitation or in vivo studies.

The Antibody Development facility occupies approximately 926 square feet on the E level of the Thomas Building on the South Lake Union site.  All necessary facilities and instrumentation required for basic tissue culture and hybridoma production in mice rats and hamsters are provided. There are sterile tissue culture hoods, Sanyo CO2 incubators equipped with a state of the art ultra violet light source to protect against contamination, Beckman GS-6K centrifuge, microfuge, analytical balance, pH meter, -20, -80, liquid N2 freezers and 4C storage space. Additionally, the hybridoma lab is well equipped for carrying out protein biochemistry to qualify test antigens, basic antibody analysis, characterization and purification techniques.

The Arnold Library supports the Center's research, education and patient care programs.  The library is centrally located in the Weintraub Building, on the Hutchinson Center’s South Lake Union Campus.  At over 5400 square feet, the library includes: a public access computer center, wireless network connectivity, study carrels, a reference collection room, book and journal stacks, a self-service copier room, an information desk, a self-checkout workstation, and work space for library staff.

Library services include course reserves, instructional support, dissertation binding, document delivery/interlibrary loan, library material photocopying, reference and literature searching, remote access (this allows library account holders to log-in and use online library resources from any computer with Internet connectivity), PubMed Central deposit and institutional repositorydeposit service, web site design and maintenance, open access journal article analysis, Center history archives, and training in the use of library resources for individuals or groups upon request.

Library resources include18,283 full-text electronic books and 23,139 electronic journals; and 5,436 hardcopy, print books.  Additional resources are outlined below: 

• Arnold Library Online Catalog – search the library’s holdings online.
• Community of Science  - Faculty Expertise Profiles, Federal Register, COS Funding Opportunities, U.S. Patents and more.
• Current Index to Statistics - An index to publications in statistics and related fields.
• Laboratory Protocols online from Springer, Wiley (Current Protocols, Cold Spring Harbor, Nature and more.
• JSTOR Mathematics and Statistics Collection - JSTOR provides access to the backfiles of 30 mathematics and statistics journal titles.
• MathSciNet – an index to journal literature in pure and applied research level mathematics from 1940 – present.
• National Library of Medicine LOCATORplus - The online catalog of journals, books, and media in the world's largest medical library.
• Ovid – Provides access to the following literature indexes, MEDLINE, Embase, Global Health, Agricola, and PsycINFO.
• ProQuest – Online, full-text general interest journals and both major and local newspapers.
• Proteome BioKnowledge Library - The Proteome BioKnowledge Library is a collection of six databases focused on the compilation of available protein information.
• PubMed from the National Library of Medicine (Get Article enabled) - NLM's version of Medline with direct links to our full-text journal subscriptions.
• Faculty of 1000 Biology – online research service that comprehensively and systematically highlights and reviews the most interesting papers published in the biological sciences, based on the recommendations of a faculty of well over 2300 selected leading researchers.  Access this directly or via PubMed.
• Web of Science
  • Science Citation Index – This index covers over 3300 science journals from 1971 to present.
  • Journal citation reports (JCR) - presents quantifiable statistical data that provides a systematic, objective way to evaluate the world's leading journals and their impact and influence in the global research community.
• Lexis/Nexis Academic - International in scope, LexisNexis academic provides full-text access to more than 6,000 legal, news and business sources.
• Science Online- Full text of the journal Science from 1880 to present, Science News, Careersand Signaling (the Signal Transduction Knowledge Environment).
• RefWorks - A Web based citation management software which may be used to gather, manage, store and share information with colleagues or generate citations and bibliographies.
• CampusGuides – A course and/or subject guide web service which library staff use to support faculty instructors and other educational activities.
• LibAnswers - A reference system & service knowledge base offering SMS/TXTing and Twitter integration.

The Biologics Production Facility supports the development and manufacture of novel biological molecules for Phase I/II clinical testing. The facility permits the reproducible production of large scale quantities of biological reagents under the strict quality control and safety conditions required by the Food and Drug Administration (FDA) for human studies. The Facility occupies approximately 2600 square feet, including a Clinical Production Suite comprised of 1300 square feet with central areas for cell production processes and purification, and related support areas for staff work space, clean staging, cold room storage and buffer preparation. All clinical manufacturing activities are conducted using approved written procedures performed by trained, qualified personnel in accordance with cGMP and other regulatory guidelines.

The Biologics Production Facility supports the development and manufacture of novel biological molecules for Phase I/II clinical testing. The facility permits the reproducible production of large-scale quantities of biological reagents under the strict quality control and safety conditions required by the Food and Drug Administration (FDA) for human studies.

The Biologics Production Facility occupies approximately 2600 square feet on the D level of the Hutchinson Building, South Lake Union site. Facility design was guided by the need to ensure adequate space and appropriate separation between primary resource activities inclusive of non-clinical production and clinical production. Segregated laboratory space, 1300 square feet total, is provided for non-clinical production and quality control. The Clinical Production Suite (CPS) is comprised of 1300 square feet with central areas for cell production processes and purification, and related support areas for staff work space, clean staging, cold room storage and buffer preparation.  The facility was constructed according to specifications set by Code of Federal Regulations Title 21, Sections 210 and 211. Validation of clean rooms and utilities were conducted in accordance with established standards.

The facility was designed to satisfy current Good Manufacturing Practices (cGMP) requirements, which are not feasible in standard laboratory settings.  Individual manufacturing suites are supplied with single-pass, class 10,000 HEPA filtered air, and all critical operations are carried out within class 100 vertical-flow biosafety or clean bench laminar flow hoods located in each area.  Unidirectional work-flow has been established so that personnel, products under manufacture, as well as equipment, components and waste follow a specific path through the suites. Qualified automation systems continuously monitor, control and alarm various aspects within the facilities, including: energy management, lab data acquisition, lighting control, access control, the status of air flow and pressurization of individual spaces within the suites, as well as the correct operation of critical equipment. 

The facility contains a range of specialized instrumentation to accommodate specific manufacturing activities. Equipment that is available for manufacturing antibodies, fusion proteins and DNA-based products allows production at scales that readily exceed the capabilities of most research laboratories, and provides the added benefit of clinical cGMP compliance that would not otherwise be available to investigators. Production is typically started as bulk cell culture in either static or spinner/shaker flask vessels, with expansion capabilities in bioreactor/fermentor formats ranging from 3 to the equivalent of 100 liter volumes. Equipment is available to harvest either bulk-conditioned media or cells depending upon project needs. In most cases bulk conditioned media is harvested followed by purification. Depending upon scale and other requirements, purification may consist of multiple steps including affinity, ion exchange, ultra filtration, and diafiltration. The resource generally takes a conservative instrumentation approach to allow for maximum product yield in conjunction with moderate capital outlay, ease in instrument repair/maintenance, minimal staffing and simplicity to facilitate FDA compliance. 

All clinical manufacturing activities are conducted using approved written procedures performed by trained, qualified personnel in accordance with cGMP and other regulatory guidelines.  Process control is in place to minimize the potential for errors or cross-contamination. At times when overlapping concurrent productions are required, segregation by types of manipulations and/or stages in the manufacturing cycle is implemented. The facility is designed to provide adequate space for manufacturing activities and appropriate separation of materials. Quality assurance is further insured by strict process control, including spatial and temporal separation of individual manipulations. Regularly scheduled cleaning of facilities and appropriate gowning of personnel ensure product safety, and contamination concerns are further minimized by the frequent use of closed production systems. 

Procedures are in place for validation of production and assay methods, development of specifications for in-process and final product release, and qualification of outside suppliers and vendors. Calibration programs, along with a comprehensive preventative maintenance plan, have been implemented for all utilities, equipment and instrumentation used for production activities.

The Cell Processing Facility provides investigators with the infrastructure and technical expertise necessary to develop therapeutic cellular products under the strict FDA regulations required for clinical evaluation. Cell production services include enrichment or depletion of specific cell subsets, provision of normal donor feeder cells, maintenance of transformed feeder cell lines, cloning, ex-vivo antigen priming, cytokine and antibody-based activation, genetic modification, expansion, cryopreservation and/or preparation for infusion of many different types of extensively manipulated cell populations. The Facility occupies approximately 4000 square feet and was designed to satisfy cGMP and other regulatory requirements.

The Cell Processing Facility supports the development and manufacture of novel cell-based therapies for Phase I/II clinical testing. The facility permits the reproducible production of large-scale quantities of therapeutic cells under the strict quality control and safety conditions required by the FDA for human studies.

The Cell Processing Facility occupies approximately 4000 square feet on the E level of the Thomas Building, South Lake Union site. Facility design was guided by the need to ensure adequate space and appropriate separation between resource activities. Segregated laboratory space is provided for quality control and other non-cleanroom support activities. Manufacturing is performed in the four dedicated suites that are designed for cell culture, material preparation, and clinical production. The facility was constructed according to specifications set by Code of Federal Regulations Title 21, Sections 210 and 211. Validation of clean rooms and utilities were conducted in accordance with established standards.

The facility was designed to satisfy cGMP requirements, which are not feasible in standard laboratory settings.  Individual manufacturing suites are supplied with single-pass, class 10,000 HEPA filtered air, and all critical operations are carried out within class 100 vertical-flow biosafety or clean bench laminar flow hoods located in each area.  Unidirectional work-flow has been established so that personnel, products under manufacture, as well as equipment, components and waste follow a specific path through the suites. Qualified automation systems continuously monitor, control and alarm various aspects within the facilities, including: energy management, lab data acquisition, lighting control, access control, the status of air flow and pressurization of individual spaces within the suites, as well as the correct operation of critical equipment such as the incubators. 

Manufacturing suites supporting the production of cellular therapeutics are equipped with biological safety cabinets and numerous double stack incubators. Other major equipment includes floor and table top centrifuges, cell washers, sterile connecting devices and large scale cell enrichment devices from Miltenyi Biotec. In conjunction with these core equipment capabilities, the quality control laboratory provides product characterization capabilities, such as automated cell counts and flow cytometry, required by the FDA for clinical studies. Every processed component is quality control tested using standardized and validated assays, with a subsequent independent quality assurance review before release for therapeutic use.

All manufacturing activities are conducted using approved written procedures performed by trained, qualified personnel in accordance with cGMP and current Good Tissue Practices (cGTP) guidelines.  The facility is designed to provide adequate space for manufacturing activities and appropriate separation of materials. Quality assurance is further insured by strict process control, including spatial and temporal separation of individual manipulations. Regularly scheduled cleaning of the facility and appropriate gowning of personnel ensure product safety, and contamination concerns are further minimized by the frequent use of closed production systems.  Procedures are in place for validation of production and assay methods, development of specifications for in-process and final product release, and qualification of outside suppliers and vendors. Calibration programs, along with a comprehensive preventative maintenance plan, have been implemented for all utilities, equipment and instrumentation used for production activities.

Collaborative Data Services provides technical support in the areas of survey implementation, quality control and data management. Services include sample development, interviewing, mailing and tracking of questionnaires, data entry, programming support, database management and respondent tracking. The resource is housed in the Arnold Public Health Sciences Building, on the Hutchinson Center South Lake Union Campus.

Collaborative Data Services provides technical support in the areas of survey implementation, quality control and data management.  Services include sample development, interviewing, mailing and tracking of questionnaires, data entry, programming support, database management and respondent tracking.

The resource is housed in the Arnold Public Health Sciences Building, on the Hutchinson Center South Lake Union Campus and shares some of its space with the Nutrition Assessment Shared Resource.  The resource occupies approximately 6,700 square feet and is readily accessible to both investigators and research project staff.  CDS is located at the Fred Hutchinson Cancer Research Center (FHCRC) South Lake Union Campus on the 1^st floor of the Arnold Building.  This square footage includes thirty eight offices or shared spaces for managers and staff, supporting three groups:   Interviewing, Data Operations and Computer Programming.

The Interviewing group has a telephone call center with nineteen privacy cubicles for interviewing and a work and storage area.  CDS has four lead interviewer offices set-up for monitoring interviewer telephones and computers for quality control.  The Data Operations group has three offices for full-time staff and four workstations for part-time employees, and a meeting area.  It also has a workroom for high-speed scanning, label printing, color printing, and mail assembly space for customized participant mailings.  The Computer Programming group has sixteen computing offices equipped with programming workstations, and a meeting area with a conference table.

The Management/Administrative Team has five management offices and two intern offices. The resource has access to several other shared spaces, including a secure short-term records storage room, a library/conference room, a break-room, a mail and copy room, and two full computer racks in a secure server room.   All spaces are in proximity to one another.

Computing and Data Collection Infrastructure

CDS utilizes a state-of-the-art computerized call center for telephone data collection.  All interviewing workstations are equipped with a computer workstation for call management and real-time data entry.  CDS builds and maintains custom software for sophisticated call management (including random digit dialing), and has also licensed DatStat Illume as a survey software engine.  All interviewing workstations can be remotely monitored by interview leads.

The software systems are maintained by CDS programmers to address the complexity and volume of research for the Consortium and launch projects in days instead of weeks.  Surveys are easily adapted and expanded without significant additional cost.  The greatly reduced time between survey design, data collection and analysis, coupled with real-time access and analysis of incoming data gives researchers the means to fine-tune surveys and respond more rapidly to new research opportunities.  The systems ensure compliance and security, support complex survey design, allow changes over time without affecting data integrity, and are compatible with sophisticated requirements—IRB and HIPAA compliance, multiple users, time periods, and languages, and integration across multiple methods, databases, and projects.  The programming team is comprised of programmers that have proven track records in custom software development.  The seasoned programming team has created databases and applications for hundreds of projects and has developed a software toolbox that they can draw upon to quickly provide custom yet affordable solutions.

CDS has a diverse mix of databases and custom applications hosted on a server infrastructure to provide hosting services for Consortium clients. The department has eight modern servers and one legacy server, all running Windows Server 2003.  CDS has four locked racks of space in two separate secure data centers in the Arnold Building.  CDS maintains primary and redundant systems, with separate servers for internal and externally accessible applications.

The CDS Data Operations group has access to the following equipment to complete their mission and to provide new services to the Consortium:  Brady Label Printer 360X (for freezer labels), Epson 4800 Color Printer and three high-throughput scanners: OpScan 8 Model 50, OpScan 8 Model 50, and OpScan 5 Model 25.

The resource offers interviewer training for scientific study personnel.  This training is performed upon request and is tailored to the needs of the specific study.  CDS has trained interview staff for English and non-English language interviews, in person and telephone-based studies, using either computer or paper-based data gathering.

In addition CDS offers several kinds of technology related seminars and courses.  CDS brown bag technology seminars are offered monthly, and cover a wide variety of software development and database administration issues.  In addition, the resource offers forty-hour software development classes once or twice a year.  Previous classes have included a master class in ASP.NET programming in C#.

Comparative Medicine provides a broad variety of animal housing, veterinary, and research support services. The facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and complies with all United States Department of Agriculture (USDA), Public Health Service (PHS), Washington State and local area animal welfare regulations. Comprehensive animal husbandry services are provided for all vertebrate animals used in the Consortium’s programs of research. All housing and use of vertebrate animals is done in Comparative Medicine’s centralized facilities which occupy approximately 50,000 net square feet.

Comparative Medicine provides a broad variety of animal housing, veterinary and research support services.  Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited; complies with United States Department of Agriculture (USDA), Public Health Service, Washington State and local area animal welfare regulations.

All housing and use of research animals at the FHCRC occurs at two independent and fully-functional sites; the Seattle Life Sciences Center (SLSC) and the Weintraub and Thomas Buildings of the Hutchinson Center South Lake Union (SLU) Campus.  Together, they offer a total of approximately 46,000 square feet of space for the animal research program. The SLU animal facilities are all located on the same floor, and are interconnected by a short corridor within Comparative Medicine's space.  The SLSC and the SLU animal facilities are separated by approximately 2.5 miles over public roads and each holds a variety of vertebrate animal species. These facilities are serviced by their own heating, ventilation, and air conditioning (HVAC) systems, but FHCRC staff is used for all animal care and most facility maintenance issues at both sites. 

The SLSC portion of the Comparative Medicine Canine Facility (approximately 12,400 net square feet) is located on the A-level of the Seattle Life Sciences Center, approximately 2.5 miles from the SLU campus. Transportation of laboratory dogs is provided via two dedicated vans that have heated and air conditioned cargo space, are maintained by the Center’s motor pool, and are driven by Comparative Medicine staff members. This site has 12 kennel-type holding rooms with a total of 82 separate enclosures for approximately 225 dogs, including areas for whelping and for quarantine of new arrivals. A separate room dedicated to canine enrichment, socialization, and training for research-related tasks is used in conjunction with the breeding program for laboratory dogs. Entry airlocks, administrative and staff support rooms, locker rooms, treatment rooms, necropsy room, a diet kitchen, food storage room, and a cage wash facility that contains a cage and rack washer, steam autoclave, and water purification system also exist at this site.  Two anesthesia machines with isoflurane vaporizers, a clinical centrifuge, an I-Stat handheld clinical analyzer, a Hematrue veterinary hematology analyzer, an ultrasonic doppler flow detector, and a veterinary dental scaler are also available for medical evaluations and support.

Animal facilities in the SLU facility contain approximately 33,702 net square feet.  Animal housing and procedure rooms comprise approximately 18,000 sq. ft. of total facility space in the SLU buildings. The Weintraub small animal facility includes a centralized cagewash/autoclave facility, a food/bedding storage room, a receiving/unpacking room for laboratory rodents, ABSL-2 biohazard containment rooms, a rodent quarantine, a research gas exposure room, a chemical carcinogen and radiation hazard containment suite, a rodent necropsy facility, a rodent surgery/procedure room, rooms devoted to laboratory rodent imaging equipment, a rodent barrier-housing and transgenic animal production facility, and a variety of housing and procedural space for laboratory rodents, Xenopus frogs, and freshwater fishes. All facilities are operated with equipment and procedures intended to maintain the animal colonies free of adventitious diseases. Only specific pathogen-free rodents are allowed in the facilities.

A broad variety of mouse and rat models, many with genetic modifications including strains with and without immune deficiencies, are accommodated via individually ventilated and HEPA-filtered microisolator cage environments (Allentown Inc.) that are autoclaved prior to use.  All animal feed and cage enrichment material is sterilized and only purified water is provided.  Husbandry is done in three-sided, height-adjustable HEPA filtered laminar flow cabinets using high-level disinfectants by gowned technicians.  Downdraft, ventilated workstations with spray-arms and halogen lighting are available at multiple locations for necropsies and tissue harvests, including perfusions with specified preservatives.  Thin client computers are supported within the vivarium so that intranet resources and research data files are always available, even when working within this barrier-like environment.

Several small animal imaging platforms utilized by the Animal BioImaging Core (ABIC) are located within the AHR Small Animal Facility so that laboratory rodents can move between technologies while protected from environmental variables, including adventitious disease, and with a minimum of physiologic stress.  Collaborative studies of the ABIC occasionally require some studies on individual tumor-bearing rodents using one imaging platform located at the University of Washington (e.g., MRI or PET) and then later on platforms located at the FHCRC (e.g, Xenogen).  Accommodation of these requirements is met in AHR through housing in negatively pressurized biocontainment-type HEPA filtered and individually ventilated cages (Allentown Inc.).  In this way they remain fully separated from all other valuable breeding colonies located concurrently elsewhere in the vivarium.  Special gowning and waste disposal procedures exist for these areas.

A transgenic production unit for the creation of transgenic and knock-out rodents via pronuclear DNA microinjection and embryonic stem cell manipulation technologies is located inside the SLU Weintraub area of Animal Health facility space. This laboratory is equipped with a Nikon Diaphot 200 microinjection manipulator, a Nikon SMZ dissecting microscope, a Sutter Instrument Co. micropipette puller, a Cellstar CO2 incubator, a Beckman TJ-6 centrifuge, a MJ Research programmable thermal cycler for PCR amplification, and -80⁰C and liquid nitrogen freezers for cell storage.

The Thomas building canine facility at SLU includes animal holding rooms with hose-down kennels or solid cages with waste catch pans, an animal diet kitchen, administrative and staff support rooms, a large chamber autoclave and laundry facility, a treatment/procedure room with self-contained intensive care unit, a dedicated sterile surgery facility with adjacent surgeon’s scrub sinks, a canine necropsy facility, and a 6 MeV 600 CD (Varian) linear accelerator which is used for total body irradiation experiments in several of the FHCRC animal-based research programs.A dedicated necropsy room fitted with down-draft table and ductless fume hood exists for post-mortem gross pathological evaluations, as required for some studies.

The Thomas Building canine sterile surgery suite contains two anesthesia machines with isoflurane vaporizers and pediatric bellows. State-of-the art intra-operative patient monitoring and support equipment in the surgery include two capnographers with pulse oximetry, a vital signs monitor with electrocardiography, and forced-air temperature management blankets.  A handheld clinical analyzer, programmable infusion pumps, several ultrasonic doppler flow detectors, and two clinical centrifuges help support patient care.  A COBE Spectra apheresis system sits in a dedicated room within the resource properly located next to a small treatment table with a dedicated circulating water blanket. All dogs are provided high quality laboratory-grade chow with supplements as needed and drinking water is delivered ad libitum via automated lixit-type water systems. A diagnostic x-ray machine with automatic film processor and a portable high-resolution laptop ultrasound unit are also located conveniently in the same area for veterinary care and research support.

The Cytokine Analysis Shared Resource provides investigators with access to state-of- the-art enzyme linked immunosorbant assays (ELISA) and other analytical methods, high-throughput equipment and the expertise to carry out a broad range of assays of human proteins. A Luminex 200 instrument provides support for cytokine analysis using microbead technology.

The Cytokine Analysis Shared Resource provides investigators with access to state-of- the-art enzyme linked immunosorbant assays (ELISA) and other analytical methods, high-throughput equipment and the expertise to carry out a broad range of assays of human proteins.  A Luminex 200 instrument provides support for cytokine analysis using microbead technology.  Currently, the resource provides over 40 assays that are available to investigators who wish to measure human cytokines, growth factors and other proteins in biological fluids. 

The Cytokine Analysis Shared Resource occupies a 750 square foot laboratory in the Thomas Building, on the Hutchinson Center’s South Lake Union Campus. One function of the laboratory is the rapid processing of both clinical and research specimens, that is, blood, serum, plasma, urine, fluid from bronchoalveolar lavage, and supernatants from in vitro experiments, using a VWR specimen refrigerator, an IRC Centra CL3R refrigerated centrifuge, a Hermle Z252MK refrigerated microcertrifuge, and a NuAire Biologic Safety Cabinet (Class II, type A/B3). Analytical equipment used for determination of cytokines, growth factors, and other biological mediators include a Molecular Devices Vmax 96-well microplate reader with Softmax Pro software linked to Macintosh computers; a Bio-Rad 1575 ImmunoWash Plate Washer; two Bio-Tek Model EL401 Manual Microplate Washers; and a Packard MultiProbe II Robotic Liquid Handling System linked to an IBM computer.  Additional laboratory equipment includes three 17 cu. ft. Forma Scientific –86 degree freezers, refrigerators, ovens, balances, water baths, pH meters, and calibrated multichannel pipettors.

The Electron Microscopy Shared resource is a full service laboratory providing transmission and scanning electron microscopy along with atomic force microscopy. The services include cryo techniques and capabilities, negative staining for high resolution viral work, particle counting, labeled antibody studies (immunogold), cell biology and structural studies. Resource instruments include two JEOL (1010 and 1230) transmission, one JEOL scanning (5800) electron microscope and one Agilent atomic force (5500) microscope.

The Electron Microscopy Shared Resource provides transmission and scanning electron microscopy and a variety of sample preparations.  Electron microscopic examination is captured using Gatan CCD cameras. Services include cryo techniques and capabilities, negative staining for high resolution viral work, particle counting, labeled antibody studies (immunogold), cell biology and structural studies.  The Electron Microscopy Shared Resource occupies approximately 2000 square feet on the E level of the Thomas Building, on the Hutchinson Center South Lake Union Campus. 

Resource equipment includes three electron microscopes supported by specialized ancillary equipment and one atomic force microscope to meet the needs of a wide range of scientific inquiry.  Electron microscopes include two transmission electron microscopes and a scanning electron microscope. A JEOL JEM-1230 transmission electron microscope is a 120 KV instrument with a Gatan 2K x 2K pixel Ultrascan 1000 bottom mount camera for high resolution work and a 11 megapixel SC1000 Orius side mount Gatan CCD camera for large field of view work. The JEOL JEM-1010 transmission electron microscope is a 100 KV instrument with a Gatan 1K x 1K pixel Bioscan 792 side mount camera.  This instrument provides back-up support and is the primary instrument utilized by resource self-users.  Both microscopes have capabilities for direct on-screen measurement of cell components, virus particles and DNA strands; low magnification capabilities to more accurately bridge the gap between light and electron microscopy; 90 degree image rotation to achieve optimal orientation of the specimen, and the ability to digitize images.  The JEOL JSM-5800 scanning electron microscope is equipped with an Orion Digital Acquisition System from JEOL. This system allows for images to be saved as standard image files, e-mail image files, or generate reports.  The atomic force microscope is the Agilent 5500 and allows for direct visualization of single molecules in solution.

The resource is also supported with Leica cryo equipment.  The equipment includes a high pressure freezing apparatus, freeze substitution system, and low temperature cryochamber for sectioning.  The equipment produces superior cellular preservation for morphological studies and better antigen retention for immunolabeling work.  Ancillary equipment includes a Ted Pella microwave, Tousimis CPD, Beckman airfuge, Denton carbon evaporator and sputter coater, two Leica microtomes, Durst enlarger and Ilford paper processor.

All computers supporting these microscopes are connected to the Hutchinson Center’s networked computer system and provide the opportunity to transfer images to remote locations within the Center.  Data storage and archiving is maintained by the Hutchinson Center’s Information Technology department.  Data is distributed via a networked computer system providing all investigators immediate access to their data.   A data management infrastructure is in place to handle data generated by the facility.  This system is scalable for expansion to meet future data storage needs.

Experimental Histopathology offers histology, special stains, immunohistochemistry, laser capture microdissection, and pathology support. We have extensive experience with human and animal tissue and provide routine support as well as customized protocol development. Immunohistochemistry services include antibody optimization, immunofluorescence (IF) and multi-color IF.

The Experimental Histopathology Shared Resource occupies 1200 square feet of laboratory space on the DE Level of the Thomas Building, on the Hutchinson Center’s South Lake Union Campus. The resource provides support for the preparation, analysis, and interpretation of tissue and cell specimens using histological and immunohistochemical methodology.  The resource has a great deal of experience working with human specimens as well as animal models.  The laboratory is well equipment with automated Tissue Tek Paraffin Processors, Tissue Tek Paraffin Embedding units, Sakura H&E stainer, Leica Microtomes, Leica Cryostats, Dako immunohistochemistry stainers, Nikon microscopes and stereoscope, and Nikon cameras. In addition, the resource is staffed with HT and HTL-certified histology technicians with a wide range of clinical and research experience.

Histology services include tissue fixation, embedding, sectioning, and hematoxylin and eosin (H&E) staining of both paraffin embedded and frozen samples. Cytocentrifugation and histogel embedding is available for preparation of cytology specimens. Special procedures have been developed to section samples for molecular analysis and laser capture microdisection. In addition, the laboratory is skilled at preparing tissue and slides for tissue microarrays (TMA). 

The laboratory offers a wide spectrum of Special Stains including periodic acid-Schiff (PAS), Wright Geimsa, Movat’s pentachrome, reticulin, silver stains, iron stain, Massons trichrome, Alcian blue, Nuclear Fast Red, and stains for various bacteria and fungus. Several techniques are available to stain whole tissue and frozen tissue sections for beta-galactosidase expression. Special stain development is also available to support the needs of various studies.

Extensive immunohistochemistry services are available. The laboratory uses automated immunochemistry stainers in order to offer consistency and standardized protocols on large and small scale projects. Over 140 hundred antibody protocols are available for a staining human and animal tissue. These antibodies include markers for cellular identification, proliferation, tumor type and progression, apoptosis, and activation. This is often made possible utilizing the antigen retrieval techniques that we have available for formalin fixed tissue. Various detection methods are offered including horseradish peroxidase, alkaline phosphatase, and fluorescence. This flexibility allows for a wide spectrum of staining including dual immunohistochemistry and multi-color immunofluorescence. The laboratory also offers services in antibody optimization, novel antibody staining protocols, and antibody validation.

Pathology review is available for histology and immunohistochemistry analysis.  In addition, the staff collaborates with a number of veterinary and clinical pathologists. The resource has a 5-headed Nikon microscope with a camera to facilitate the review of samples and capture critical data.

The Flow Cytometry lab occupies roughly 1800 square feet on the E level of the Thomas building on the South Lake Union campus. The facility provides researchers access to seven cell analyzers and eight cell sorters. Detection capabilities on these instruments range to five excitation lasers and eighteen color emission. The facility provides a number of services including: training on equipment use, assistance with experimental design, and consultation on data analysis and interpretation.

The Flow Cytometry Shared Resource provides support for cell analysis and sorting.  The resource occupies roughly 1800 square feet on the E level of the Thomas Building, at the Hutchinson Center’s South Lake Union campus.  Included in the facility are dedicated rooms for cell sorters, common rooms for benchtop analyzers, a wet lab, offices, and storage. 

The facility maintains five fluorescence activated cell sorters, seven benchtop cytometers, and two magnetic bead sorters.  Three of the cell sorters are Becton Dickinson FACS Aria II cell sorters.  One of these is equipped with five lasers and can detect up to 18 colors.  A second Aria is equipped with four lasers and can detect up to 13 colors.  The last Aria is equipped with three lasers and can detect up to 18 colors.  The facility has a Becton Dickinson Influx high speed cell sorter that is equipped with five lasers and can detect up to 15 colors.  The fifth sorter is a Union Biometrica Copas 250 large particle sorter equipped with two lasers and three detectors.  Two of the Bench top analyzers are Becton Dickinson LSR II cytometers.  The first being equipped with five lasers and capable of detecting up to 18 fluorescent markers, while the other is equipped with four lasers and detection of up to 18 markers.  Both of the LSR IIs are equipped with High Throughput Samplers (HTS) capable of running 96 samples in as little as 15 minutes.  The facility has a three laser 8 color Becton Dickinson Canto II also equipped with an HTS unit.  The facility also maintains a Becton Dickinson FACS Canto analyzer capable of detecting up to 6 fluorescent markers.  Additionally, the laboratory has three Becton Dickinson FACS Calibur cytometers that are equipped for four-color detection.  One of these Caliburs is also equipped with a high throughput plate reader.  The flow cytometry laboratory also operates two Miltenyi AutoMACS Pro magnetic bead cell sorters one of which is dedicated for sorting cells for long term storage, while the other is for general use.

Data storage and archiving is maintained by the Center’s Information Technology department.  Data is distributed via a networked computer system providing all investigators immediate access to their data.   A data management infrastructure is in place to handle data generated by the facility.  This system is scalable for expansion to meet future data storage needs.  The flow cytometry facility supports a variety of desktop software which is available to all users, facilitating interactive analysis on investigator’s laboratory computers.  Remote data analysis includes Cell Quest, WinList and FloJo software applications for general analysis, and Multicycle AV and FloJo for cell cycle modeling. 

After hours access to the lab and instrumentation is dependent upon user completion of training course certification.  The lab offers informal one-on-one or small group training to researchers on the proper use of both the cell sorters and benchtop analyzers.  Informal and on-line training is available on the use of lab supported flow cytometry software packages, including CellQuest, CellQuest Pro, FACS Diva, FloJo and Multicycle.

The Genomics Shared Resource has state-of-the art instrumentation and experienced staff who provide microarray, CE sequencing, NextGen sequencing, and RNAi screening services. Technologies include: Affymetrix, Agilent, Illumina, and NimbleGen (microarrays); ABI/Life Technologies (CE sequencing and qPCR); Illumina HiSeq 2000 (NextGen sequencing); and various shRNA and siRNA libraries from Open Biosystems, Qiagen, and Invitrogen (RNAi). The Resource also has sophisticated data management and analysis resources, as well as the expertise to assist in data processing and analysis.

The FHCRC Genomics Resource provides services and support to the genomics-based research needs of FHCRC.  The Resource is comprised of the Genetic Analysis Laboratory, providing DNA sequencing, genotyping, and quantitative (real-time) PCR services; the DNA Array Laboratory, providing expertise in numerous microarray technologies and applications; and a developing laboratory supporting RNA interference (RNAi) technologies.

Genetic Analysis Laboratory

The Genetic Analysis Laboratory occupies 705 sq. ft. on the E-level of the Thomas Building, FHCRC South Lake Union Campus.  The lab offers capillary-based and NextGen sequencing services.  The laboratory also provides access to user-operated equipment for real-time quantitative PCR.  Lab staff processes sequencing reactions using a dedicated Applied Biosystems 3730XL DNA Analyzer.  A second 3730XL DNA Analyzer is reserved for SNPlex and fragment analysis applications.

NextGen sequencing technology includes a state-of-the-art, high-throughput sequencing-by-synthesis (HT-SBS) Illumina Genome Analyzer IIx (GAIIx) System.  In Fall 2010, the lab will add an Illumina cBot cluster station and a HiSeq 2000 sequencer.  Support equipment includes a Covaris S2 System used in the preparation of sequencing libraries, as well as an ABI StepOne Real-Time PCR System for use in library QC. 

NextGen sequencing services are obtained through a module of HutchBASE, a customized version of BASE v2.6 (BioArray Software Environment) that was originally developed at Lund University in Sweden.  In addition to sample annotation and tracking features, the HutchBASE LIMS also automatically triggers data analysis pipeline protocols, produces data QC reports, and provides data management capabilities.

The Genetic Analysis Lab also houses four user-operated ABI Prism® 7900 Sequence Detection Systems, one of which is fitted with an automated robotic loading module for increased screening throughput capabilities.  Lab members work with researchers by providing training on these instruments.  FHCRC researchers can access relevant processing and analysis software through an IP-restricted website (http://genomics.fhcrc.org/).

DNA Array Laboratory

The DNA Array Laboratory occupies 1900 sq. ft. and is located on the E-level of the Thomas Building, FHCRC South Lake Union Campus.  The lab was developed in 1998 and offers researchers access to four different commercial microarray platforms: Affymetrix GeneChip, Illumina BeadChip, NimbleGen, and Agilent Technologies. Resource personnel perform all functions pertaining to microarray technology, providing consultation on experimental design, approaches to troubleshooting, and assistance with data quality and analysis issues.

The laboratory routinely performs Affymetrix GeneChip expression and SNP mapping services, which include assistance with experimental design, sample QC testing and troubleshooting, hybridization and scanning services, analysis software training, and data analysis consulting.  The laboratory houses a complete Affymetrix GeneChip system, including four GeneChip model 450 fluidic stations, two GeneChip model 640 hybridization ovens, and a GeneChip 3000 7G scanner with autoloader.  The laboratory also maintains Affymetrix GeneChip Command Console (AGCC), Expression Console, and Genotyping Console software packages, which are used to process and analyze GeneChip array data.

In addition, the laboratory has an Illumina Beadstation System, including one BeadArray Reader, one iScan Reader, and additional supporting automation (chip autoloader and a Tecan liquid-handling robot).  The system is capable of handling the full spectrum of Illumina assays:  Infinium Whole-Genome Genotyping Assay, GoldenGate (custom-designed) Genotyping Assay, Methylation Profiling Assay, Direct Hybridization Gene Expression Assay, and DASL Gene Expression Assay. 

General laboratory equipment includes, 12 Applied Biosystems 9700 PCR thermocyclers, a NanoDrop UV/Vis spectrophotometer, and two Agilent 2100 "Lab-on-a-Chip" Bioanalyzers.  Hybridizations are typically performed using one of the lab’s four Maui Hybridization Stations. The lab also has two Robbins Scientific Hybridization Incubators for use with Agilent Technologies arrays.  Post-hybridized array images are collected using one of the facility’s two Axon Instruments GenePix 4000B microarray scanners, which are housed in a SciGene NoZone Workspace.  The workspace includes an ozone ‘scrubber’ and an ozone monitor that are interfaced with a Plexiglas enclosure.  The laboratory also has a high-resolution Agilent Technologies DNA Microarray Scanner.

Microarray data management and analysis support is provided by Resource personnel. Microarray data is stored and accessed via HutchBASE, a customized version of BASE v2.6 (BioArray Software Environment) that was originally developed at Lund University in Sweden.  BASE is an open-source MIAME compliant comprehensive web-based data storage and handling solution with additional LIMS capabilities.  It is used for sample submission, order processing, array inventory, and workflow tracking, as well as data QC, pre-processing, and normalization.  The application also allows for data interfacing with a number of downstream analysis applications.  Data analysis and annotation tools provided by the Resource include commercial software (e.g., Partek Genomics Suite, Ingenuity Pathway Analysis), as well as open-source software such as SAM, Cluster/TreeView, CyberT, FatiGo, BioConductor packages (e.g., limma), MeV, UCSC Genome Browser, etc.  Data management and analysis tools are available to researchers through an IP-restricted website (http://genomics.fhcrc.org/).  This website includes software downloads/connectivity instruction, instructional and training documents, and supporting files relevant to each application. 

RNA Interference (RNAi) Laboratory

The RNAi Lab occupies two areas on the C2-level of the Hutchinson Building, FHCRC South Lake Union Campus.  One of these areas is 380 sq. ft. and consists of a wet lab adjacent to a storage area that houses two -80°C freezers holding Open BioSystems’ Human and Mouse GIPZ lentiviral shRNAmir clone collections.  Each collection consists of approximately 65,000 unique shRNA clones.  The storage area also holds a -20°C freezer containing Qiagen’s Human and Mouse Druggable Genome and (custom designed) Transcription Factor siRNA sets.  Support equipment in the lab includes a dual-arm Beckman-Coulter BioMek FX with 96-channel and vari-span 8 channel pipette heads and includes a 160-plate stacker carousel with barcode scanner, a Molecular Devices’ Spectromax Plus absorbance microplate reader, a Genomics Solutions HiGro Microwell Plate Growth System, and other general laboratory equipment.
 
An additional 140 sq. ft. lab is located in proximity and houses equipment to facilitate single-well siRNA screens.   Equipment includes two Napco 8000DH C02 incubators, a BioTek ELx405 plate washer with a 50 plate BioTek Plate Stacker, a BioTek Microflo Dispensing System with a 50 plate BioTek Plate Stacker, and a Perkin Elmer EnVision Multi-label Plate reader that includes a barcode reader and a 50 plate stacker for automatic loading. 

RNAi Screening Services are obtained through a module of HutchBASE, a customized version of BASE v2.6 (BioArray Software Environment) that was originally developed at Lund University in Sweden.  In addition to sample annotation and tracking features, the HutchBASE LIMS also automatically triggers data analysis pipeline protocols, produces data QC reports, and provides data management capabilities.

Glassware Services is responsible for cleaning and sterilizing reusable laboratory glassware on a timely basis.  Procedures are routinely available to all laboratories on the Hutchinson Center’s South Lake Union Campus and include washing, drying, and sterilization of glassware, pipette plugging and autoclaving of laboratory liquids.  Designated personnel within the facility are responsible for processing and sterilization of biohazardous waste from all laboratories.  The resource maintains its College of American Pathology accreditation. 

The facility occupies 2929 square feet in the Weintraub Building and 600 square feet in the Thomas Building (for the processing of biohazardous waste).  The central glassware facility is equipped with washers, dryers and ancillary support equipment.

The Immune Monitoring Lab provides investigators with a broad range of services and expertise where precise evaluations of the immunologic response are required. Resource services include the production of peptide-MHC Class-I tetramers, unique reagents for accessing antigen-specific T cells; molecular assays for T cell receptor related analysis, cellular assays for determining cellular immune functions.

The Immune Monitoring Laboratory (IML) support investigators with resources and expertise to evaluate cellular immunologic processes and responses.  The IML provides reagents, molecular and cellular assays, assay development support and research consulting services. These include peptide-MHC tetramers, TCR spectratyping, QPCR tracking assay for TCR, TREC assay, immunophenotyping by flow cytometry, cytotoxic and lymphoproliferation assay and NK/LAK assay. In addition the resources provides instrumentation support and technical trainings.

The IML is located at the E level in the Thomas Building and occupies approximately 700 square feet space including the lab, office and a equipment room.  All necessary instrumentation required for processing and analysis are provided. Equipment includes a biosafety cabinet, a CO2 incubator, a FPLC system, an ELISPOT reader, an ELISA reader, a QPCR system, a thermocycler, a top-Count, a cell harvester, a luminometer, a microplate washer, two larger and two micro-centrifuges, a -80°C freezer, a -20°C freezer/refrigerator combination, two -20°C freezers, a refrigerator, a 4°C deli-case, a liquid nitrogen tank, two microscopes, a water bath, a shaker incubator, two balances, a spectrophotometer, a cell counter and other small instruments, such as pH-meter, heat blocks, vortex mixers, stirrers, picofuges, pipettes.

Service description

pMHC tetramers are custom-made reagents which bind to antigen-specific T cells receptors. Tetramers are produced by refolding of recombinant MHC proteins and designated epitopic peptides. We have the commonly studied MHC Class-I proteins from human, mouse and macaques. We provide monomers, fluorochrome-conjugated tetramers and Qdot-conjugated multimers.

Spectratyping is a set of multiplex RT-PCR reactions that amplifies all T-cell receptor beta families to reveal the clonal length polymorphism in the CDR3 region. This technique is used to access clonal composition in T-cell populations. Specifically, it is often used to determine clonal expansion, contraction and deletion during immune response and reconstitution. TCR spectratyping can be provided for PBMC samples or for FACS-sorted cell subsets.

The unique feature of each T-cell clone is found in the recombined T cell receptor sequences. IML can identify the clonal TCR beta chain sequence by RT-PCR and direct sequencing of the clonal cell sample. The clonal TCRB sequence can be cloned and used for QPCR analysis. With knowledge of the clonal TCR sequence, specific primers and a probe for quantitative real-time PCR analysis can be designed, allowing the quantitation of the clonal T cells in a cell population. We provide this service in the setting of T cell adoptive transfers in immune therapy trials of cancer patients.

TREC is a quantitative real-time PCR analysis of the TCR excision circle, a circular DNA fragment excised from the genome as a result of the TCR rearrangement in the thymus. This circular DNA is not capable of replication and is therefore diluted as T cells divide in the periphery. The relative quantity of the TREC DNA provides a measurement of thymic output of naïve T cells.

The Lymphoproliferation assay is the classic method for assessing lymphocyte response to stimulation. Lymphocytes proliferate after incubation in vitro with mitogens and specific antigens, or in a mixed lymphocyte reaction, which is then measured by 3H-thymidine incorporation. The rate of proliferation provides an indication for general lymphocyte activity or responsiveness to specific antigens. The Immune Monitoring resource has provided this assay for several clinical trials in accordance to specific study requirements.

Cytotoxic T cells (CTL) and Natural Killer (NK) cells function as their names indicate by killing their target cells, which can be measured by the 51Cr - release assay. Specific target cells are loaded with Chromium-51, which is then released into the medium when the cells are lysed by specific CTL or NK cells. This functional assay is widely used in studies of antigen-specific CTL and NK activity.

Analyzing cell surface and intracellular proteins by labeling with fluorochrome-conjugated antibodies and flow cytometry is a common method in immunology. Flow cytometric analysis can be used to enumerate cell subsets according to their differential markers, activation, functional and other characteristics. Specific cell subsets can be sorted out from a large population of cells using a cell sorter. Antigen-specific T cells can be analyzed after labeling with specific pMHC tetramer. The Immune Monitoring lab provides cellular analysis by flow cytometry in accordance to specific study requirements. For example, we have provided complete peripheral lymphocyte phenotyping of B cells, T cells, Monocytes, NK and dendritic cells for the evaluation of immune reconstitution after stem cell transplant.

The Enzyme-linked ImmunoSpot (Elispot) assay is a high-throughput procedure to evaluate antigen-specific T cell responses on a single cell basis. In this assay, cells that are responding to antigenic stimulation secrete cytokines which are captured locally by antibodies, and visualized by staining in the form of spots. Thus, precise enumeration of responsive cells can be made. The Immune Monitoring resource provides training and technical support for investigators performing this assay and using the Elispot reader. We also provide complete assays of clinical cell or blood samples.

The Nutrition Assessment Shared Resource (NASR) provides state-of-the-art methods for collection of dietary intake data from participants in observational and intervention studies. The resource supports collection and analysis of many dietary assessment instruments.

The Nutrition Assessment Shared Resource (NASR) provides state-of-the-art methods for collection of dietary intake data from participants in observational and intervention studies.  The resource supports collection and analysis of many dietary assessment instruments. 

The resource is physically located in the Arnold Building on the Hutchinson Center’s South Lake Union Campus.  The shared space includes a telephone monitoring room, small conference room, work room, printer stations, break room and kitchen, scanner and mail room and storage room.   The shared interviewer space is approximately 1500 square feet and consists a general administrative area and 19 interviewer cubicles each set-up with a high-speed computer, monitor, keyboard, phone, headset and desk space.  This shared space is sound-proofed and used by NASR for 24-hour dietary recall interviews and data entry of food records.   Approximately 600 square feet of office space is provided to house resource personnel.  Resource support equipment includes a copy machine, printers, a fax machine, National Computer Systems OpScan 5s and other standard office equipment.

The Prevention Center conducts study interviews, collects medial history and physical exam data, conducts classes with study participants, supports biospecimen collection, indirect calorimetry and endoscopy. The 12,560 square foot Prevention Center includes facilities for clinical and anthropometric measurements, exercise testing and training, and feeding studies, each located on the E-level of the Arnold Building at the Hutchinson Center’s South Lake Union Campus.

The Prevention Center conducts study interviews, collects medial history and physical exam data, conducts classes with study participants, supports biospecimen collection, indirect calorimetry and endoscopy. 

The 12,560 square foot Prevention Center includes facilities for clinical and anthropometric measurements, exercise testing and training, and feeding studies, each located on the E-level of the Arnold Building at the Hutchinson Center’s South Lake Union Campus.  The 5,274 square foot research clinic includes a dedicated, full time receptionist, a check-in and waiting area for all research volunteers,, 15 exam rooms, a DEXA scanner room, 4 interview rooms, 3 restrooms  for volunteer use only, a pharmacy, a classroom, a phlebotomy area, a processing lab with hood capability and specimen pass-through from an attached private restroom. Two exam rooms are outfitted for gynecologic capabilities including an electronic adjustable table for volunteers with limited mobility.

The Prevention Center includes a 3,978 square foot exercise testing and training facility with state-of–the-art equipment for performing graded exercise testing (exercise tolerance testing, VO2max testing).  It includes a MedGraphic Cardio2 metabolic cart coupled with a Trackmaster treadmill.  A code cart is present and fully supplied with medical response medications and supplies, and staffs are trained regularly with code drills.  The training facility includes 7 PRECOR treadmills, 2 Quinton treadmills, 5 PRECOR recumbent bikes, 3 PRECOR Elliptical Trainers, 2 Concept II rowing ergometer and 6 CYBEX strength-training machines that work all of the major muscle groups.  There are male and female shower facilities with built in emergency response systems..  It additionally contains a 400 ft2 multi-purpose room to be used for health and wellness research and education activities.  Both the training facility and the multi-purpose rooms are set up with the latest audio-visual technology to enhance activities with video and audio components.

The Human Nutrition Lab (HNL) has dedicated space in the Arnold Building on the E level within the Prevention Center, convenient to parking, phlebotomy and reception.  The new facility is 2,908 square foot and includes extensive food storage, preparation, and service areas.  Over a third of the square footage is devoted to storage, allowing the purchase of food in lot-size amounts, and includes a walk-in freezer, refrigerator, and dry storage. The food preparation facility includes both cold and hot food preparation and production areas with a gas cooktop, convection ovens, steamers, microwave oven, blast chiller, Mettler-Toledo balances, and heat-sealer. The volunteer cafeteria includes a tray assembly/service area and dining room.  The service area includes a serving station, double door pass-through refrigerator, roll-in refrigerator, holding cabinet, 4 microwaves for reheating meals, and beverage station.  The dining room seats up to 36 at one time and can be used for classes and other group activities as necessary.  A networked computer system is used for data entry, diet development, and HNL food preparation documents.  Available nutrition computer software includes ProNutra and NDS-R.

The Proteomics Shared Resource provides mass spectrometry and HPLC services for the analysis of proteins and peptides. Mass spectrometry services include molecular weight determinations of proteins and peptides, protein identification and quantification, and protein modification characterization. Instrumentation includes Thermo Electron LTQ-FT, LTQ, LTQ OrbiTrap, TSQ Vantage and LCQ DECA-XP. HPLC services include protein and peptide purification and fractionation. Sample preparation services are also available which include proteolytic digestion of proteins in gel slices and solution and sample desalting and concentration.

The Proteomics Shared Resource provides support for mass spectrometric analysis including MALDI mass analysis, LCI ESI mass and tandem analysis.  The Proteomics facility is located at the Hutchinson Center’s South Lake Union Campus on the E level of the Thomas Building.  Combined “wet” bench and instrument space consist of 2,100 sq. ft.  Additional space is provided for the Proteomics computer cluster and office space for resource personnel.

Services Provided
Sample Preparation: The facility provides expertise in preparing samples, primarily proteins and peptides in subpicomole quantities, for mass spectrometric analysis.  Services include enzymatic digestion of proteins in solution or from gel slices and sample desalting and concentration.  HPLC purification is available for the purification and/or fractionation of proteins and peptides via ion-exchange and reverse phase separations.  Isolation and enrichment of phosphopeptides via metal-based affinity techniques is also an available service.  Consultation with the facility staff is encouraged for experimental design considerations, such as estimating protein quantities needed for analysis and identifying contaminants that might inhibit analysis. 

LC-ESI-MS:  Protein identification is provided for purified proteins or complex mixtures of proteins.  Prepared samples are separated by liquid chromatography (LC) and peptides are directly eluted into an electrospray ionization mass spectrometer.  Tandem mass analysis of the peptides provides data for database searching with X!Tandem to identify the proteins present.  Tandem mass analysis is also used to identify sites of protein modifications.   

Five liquid chromatography electrospray ionization (LC ESI) mass spectrometer systems are operated by the facility.  All five systems use Eksigent NanoLC-2D autosamplers and HPLCs connected to either ThermoElectron LTQ, LTQ-FT, LTQ-OrbiTrap (x2), or TSQ Vantage mass spectrometers.  All of the systems are automated for use 24 hours per day, 7 days per week and the HPLCs are configured for flow rates of less than 500 nanoliters per minute and use columns that are less than 100 microns in internal diameter.  The detection limits for peptides is about 200 attomoles for all three systems.  With the exception of the TSQ Vantage, these systems are primarily used for identifying proteins and characterizing post-translational modifications on proteins and peptides, while the LTQ-FT and the LTQ-OrbiTrap are also used for global quantitative experiments (SILAC, d0-d3 acrylamide labeling).  The TSQ Vantage is used for highly selective (targeted) and highly sensitive qualitative and quantitative assays for peptides and small molecules.

MALDI & LC-MALDI Mass Analysis:  The facility operates a matrix-assisted laser-desorption ionization tandem time-of-flight mass spectrometer (MALDI TOF-TOF), along with a LC-MALDI spotting system.  The MALDI TOF-TOF instrument is an Applied Biosystems 4800 Proteomics Analyzer capable of performing tandem MS (peptide sequencing) experiments.  This instrument is used to support protein sequence mapping to determine sites of protein modifications, protein identification, partial de novo sequencing of peptides, protein profiling for biomarker discovery, and molecular weight measurements of proteins, peptide, and oligonucleotides.  The 4800 mass spectrometer has a limit of detection for peptides of about 200 attomoles.  The LC-MALDI system consists of an Eksigent NanoLC-2D HPLC coupled in-line with a LC Packings Probot spotting station.  This system is primarily used to purify and separate peptide mixtures and then directly spot the separated peptides onto a 4800 MALDI target for analysis.

Mass spectrometry data are analyzed by software called X!Tandem to compare mass spectrometry data to protein databases for the identification of proteins.  X!Tandem is designed for parallel computing and it is used on a 120-processor computer cluster.  Software called XPRESS and Q3 are used to obtain quantitative information from mass spectrometry data.  Qualitative and quantitative results are stored in a SQL relational database called CPAS (J. Proteome Res. 5:112-121 (2006)) and results can be reviewed via a web-based viewer.  Various tools have been built into the viewer to assist with reviewing the results, such as filtering, sorting, and comparing results, and protein annotations are linked to the identified proteins to provide rich biological information.  Assistance with data analysis is provided by the resource staff as well as the developing computation resource at the Center.

Data storage and archiving is maintained by the Center’s Information Technology department.  Data is distributed via a networked computer system providing all investigators immediate access to their data.   A data management infrastructure is in place to handle data generated by the facility.  This system is scalable for expansion to meet future data storage needs.

The Proteomics Shared Resource periodically offers a week-long course on mass spectrometry-based proteomics.  The course covers general principles of protein separation, mass spectrometry, and qualitative and quantitative proteomics.  Data analysis is also covered and participants are introduced to the available data analysis tools.
 

The Scientific Imaging Resource serves as a centralized facility for optical microscopy, gel and blot imaging, and for the visualization and quantitative analysis of gel, blot and microscopy data. The facility provides access to eight fluorescence microscopes, including laser scanning spinning disk and swept field confocal microscopes, a Deltavision fluorescence deconvolution microscope, a high content analysis system for automated imaging an analysis of fluorescent samples in multi-well plate format, a digital pathology system for automated scanning of stained tissue sections and tissue microarray slides, a total internal reflection microscope (TIRF) and a general purpose microscope for transmitted light and fluorescence image capture. Gel and blot imagers include a GE Typhoon Trio and a Li-cor Odyssey near infra red scanner. Several image analysis software packages are available for data visualization and quantitative image analysis.

The Scientific Imaging Shared Resource provides support with a variety of optical microscopy technologies, gel and blot imaging, visualization of multi-dimensional microscopy data, and quantitative image analysis. The Imaging facility occupies approximately 1800 square feet of laboratory space, and is located at the Hutchinson Center’s South Lake Union Campus on the E level of the Thomas Building.

Services provided.

Light Microscopy. The resource provides state of the art instrumentation and support for the imaging and analysis of biological samples at the light microscope level, including brightfield (transmitted light) microscopy, fluorescence microscopy, 3D image restoration (deconvolution), single point laser scanning and swept field confocal microscopy, automated plate imaging and analysis (high content analysis), whole slide scanning and analysis (digital pathology), multi-photon microscopy, and advanced techniques such as fluorescence recovery after photobleaching (FRAP), photoactivation, and fluorescence resonance energy transfer (FRET). Live cell and time-lapse microscopy are available on several of the resource's instruments.
Gel and blot imaging. The resource provides two dedicated scanners for the imaging and quantitative analysis of fluorescent, chemiluminescent, and radioactive assays in gels, blots and microplates. A professional flat bed scanner is also available for digitization and quantitation of conventional photographic film (autoradiograms). Instrumentation available within the resource is listed below:

Applied Precision, Inc. Deltavision Wide-field Deconvolution Microscope.
The Deltavision system uses a computational approach based on Fourier transform mathematics to remap the out-of-focus components of three-dimensional fluorescent images. By using a computational approach coupled with cooled CCD detection, this system is able to achieve outstanding sensitivity and resolution. In addition, since the system is not laser based, a wide range of excitation and emission wavelengths can be employed (350nm to >700nm. Fluorescence filters are available for the imaging of a wide range of fluorescent molecules including fluorescent proteins. The system is ideal for high resolution imaging of cells, and for time lapse microscopy.

Zeiss LSM 510 Confocal Microscope
The Zeiss LSM 510 META confocal and two photon microscope offers three visible range lasers with major excitation lines at 456/488, 543, 633 nm, and is capable of imaging up to eight fluorescent channels.  This instrument is also equipped with a Coherent Chameleon ultrafast pulsed IR laser for multiphoton microscopy. This laser is continuously tunable in the 720-930 nm range, and allows the excitation of a variety of fluorescent dyes, including UV-excited dyes such as DAPI. The system includes devices for temperature control, and is well-suited for the imaging of live cells or embryos.  The Zeiss 510 is equipped with the spectral META detector, which allows the separation of dyes with closely related emission spectra. Finally, image acquisition can be automated, for the sequential imaging at multiple locations.  The system includes top-of-the-line optics for most imaging needs, and a number of automated features (tiling, multi-point visiting, FRAP) available.

Nikon TiE swept field confocal and widefield fluorescence microscope (Nikon Live).
The Nikon Live is a versatile microscope system for time lapse imaging of living samples. The system includes a Prairie Technologies Swept Field module for fast confocal imaging of samples labeled with CFP, GFP, YFP and RedFP, and similar fluorescent reporters. The system also allows conventional (widefield) fluorescence and transmitted light imaging, including phase contrast and DIC. The system is equipped with a fast piezo stage and motorized filter wheels for fast acquisition of multi-channel 3-D image stacks. The system includes an environmental enclosure to provide controlled temperature and CO2. The microscope is equipped with a very sensitive Photometrics QuantEM EMCCD camera for confocal imaging, and a high resolution Photometrics Coolsnap HQ2 for conventional fluorescence and transmitted light imaging.

Cellomics Arrayscan VTI High Content reader.
The Cellomics Arrayscan is a dedicated microscope for automated imaging and analysis of fluorescent samples in multi-well plate format. The system is suitable for high throughput image-based screens. The instrument is equipped with three hotels and a Catlyst Express robotic arm for unattended loading of up to 45 plates. The system also includes Cellomics High Content Informatics software to extract quantitative data from plate images. Data is stored in a database format on a 2 TB server for viewing and analysis.

Tissuegnostics Tissuefax Digital Pathology system.
The Tissuefax is a semi-automated microscope system for the scanning and digitization of pathology slides, including large tissue sections and tissue micro-array (TMA) slides. The system includes a motorized stage and autofocus for semi-automated processing of up to eight slides per session. The system is capable of true-color image acquisition for transmitted light samples (such as immunohistochemistry slides), and a monochrome camera for acquisition in fluorescence mode (Immunofluorescent samples) of up to four channels.The system also includes TissueQuest and Histoquest software for quantitative analysis of Tissuefax data.

Nikon E800 fixed-stage fluorescence microscope.
For routine examination of samples in transmitted light and fluorescent mode, the Nikon E800 provides straightforward operation, high quality optics, and imaging versatility with dual CCD cameras for fluorescence and transmitted light work, allowing acquisition in grey scale and color modes. The microscope is equipped with DIC optics for the imaging of unstained specimens, and is equipped with filters for the detection of major fluorescent dyes, including DAPI, Fluorescein, GFP, Rhodamine, Texas Red, and Cy5. Acquisition of single frames, or time lapse series, is done with Metamorph software.

Typhoon Variable Mode Imager
The facility is equipped with a GE Typhoon Trio scanner for the imaging and analysis of radioactive, fluorescent and chemiluminescent samples.  Phosphor screen technology allows sensitive detection of radioactive samples. With five orders of linear dynamic range, much higher than conventional photographic or X-ray film, the Typhoon can capture an image from both strong and weak radioactive signals using a single exposure.  Equipped with three lasers and a set of emission filters, the instrument allows the fast and quantitative, analysis of Southern, western, and northern blots using fluorescent (non-radioactive) techniques. The instrument is also capable of imaging chemiluminescent samples. A multi-user license for ImageQunat TL (IQTL) is available, and allows users owning Windows PCs to analyze and quantitate gel data in their own labs.

Li-Cor Odyssey scanner.
The Li-Cor Odyssey is a dual channel scanner for the detection of far red/ infra-red fluorescence in gels, blots, and multi-well plates. This instrument offers high sensitivity, low background, and is a simpler and more quantitative alternative to chemiluminescent detection. It is particularly recommended for protein studies. Dedicated proprietary Odyssey software is available for data analysis.

In addition to the primary instruments, the resource operates four imaging computer workstations for the visualization, modeling, and quantitative analysis of large datasets such as high resolution 3-D confocal stacks or time-lapse sequences. These workstations run advanced imaging software, including Volocity, Imaris, Zeiss LSM confocal software, Metamorph, ImageQuant TL, Cellomics Bioapplications, as well as public domain Image J.

Data storage and archiving is provided by the Center’s Information Technology department.  Data is distributed via a networked computer system providing all investigators immediate access to their data.   A data management infrastructure is in place to handle data generated by the facility. This system is scalable for expansion to meet future data storage needs.

Specimen processing provides a broad range of services including the development and validation of procedures for specimen processing analyses; processing of blood, urine and DNA samples and DNA extraction. Total facility space is 1,350 square feet; laboratory equipment includes tissue culture hoods, nine centrifuges, four bench top incubated shakers, freezers, refrigerators, spectrophotometer, fluorimeter, and ancillary support equipment. A networked computer system provides access to systems operated to support population based studies which is used for case ascertainment, data entry and data base management.

The Specimen Processing Shared Resources provides a broad range of services including the development and validation of procedures for specimen processing analyses; processing of blood, urine, buccal and DNA samples and DNA extraction.

Resource operations are split into three sites, for a total of 1,451 net square feet.  Space is provided on the C level of the Hutchinson building, 5th floor of the Arnold Building at the Hutchinson Center’s South Lake Union Campus, and a satellite lab is located on the 7th floor of the Seattle Care Alliance. All necessary facilities and instrumentation required for processing and tissue culture activities are provided.  Equipment within the laboratories include fluorometer, spectrophotometer, automated cell counter, sterile tissue culture hoods, fume hoods, centrifuges, freezers, refrigerators and ancillary support equipment.   A networked computer system provides access to a system operated to support population based studies which is used for case ascertainment, data entry and data base management.