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Core Research Facilities
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| Core Gene Expression & Proteomics Module |
Imaging Module |
| Hybridoma Module |
Molecular Biology Module |
| Morphology Module |
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 Core Gene Expression & Proteomics Module
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The effect on human host cell gene expression of viral infection; while viruses are relatively simple biota their subversion of the host cell may cause complex and unexpected changes in host gene expression.
The effect of different growth factors on cells cultured from the corneal stroma. We have found that our microarray data accurately reflects some known effects of TGF-b1, i.e., immunosuppression, increased elaboration of extracellular matrix, and increased synthesis of cytoskeletal components. We are now examining the expression changes which control cell growth and intracellular signaling.
The effect of specific signal induction inhibitors on expression in corneal cell subtypes. For many genes, expression is likely controled by inputs from multiple signaling pathways. Identifying the small but consistent changes which occur when these individual pathways are inhibited will allow us to assemble a map of cellular control circuits.
Current projects use predominantly Affymetrix GeneChip DNA microarrays. Microarray sample preparation and data analysis are performed within the Module, while samples are scanned in the Department of Pathology. An ABI 7700 instrument is used to perform real-time PCR, both for confirmation of microarray data and to assay the expression of genes not available on GeneChips.
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| Contact Information | Stephen Harvey
Ph.D.
412-647-9255
sah@pitt.edu
EEINS-911, 203 Lothrop Street, Pittsburgh PA 15213 |
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Imaging Module
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Director: Nirmala SundarRaj, PhD
Purpose
The Electronics/Molecular Imaging module is dedicated to providing hardware and software support for acquisition, analysis and storage of digital microscopic images.
Laser Scanning Confocal Microscopy
Laser scanning confocal microscopy addresses several of the problems encountered in traditional microscopy by controlling the depth of filed, eliminating image degradation through out-of-focus information and by allowing the collection of serial optical sections from thick specimens.
Our confocal system, an Olympus Fluoview1000 is equipped with an Olympus IX81 inverted microscope, offers mercury lamp excitation for initial viewing and four lasers with six possible wavelengths of laser excitation for confocal acquisition. It has 10x, 20x and 60x air objectives with phase contrast available as well as 20x, 40x and 60x and 100x oil objectives.
Live Cell Microscopy
Live cell microscopy allows researchers to observe and quantify phenomena in living cells via technological advancements in hardware, software and laboratory techniques. This approach to microscopy requires creation of an environment hospitable to maintenance of cell life for the duration of the experiment.
Our live cell system revolves around the Nikon Eclipse TE200-E inverted microscope with 4x, 10x and 20x air objectives and 40x and 60x (TIRF) objectives. Illumination is provided by a DG-4 xenon light source and a wide variety of excitation and emission filters are available. Peripherals include an Eppendorf microinjection system, a Roper CoolSnapHQ camera, uniblitz brightfield shutter, a heated objective, stage and chamber cover, x, y and z automated stage control and a suspension table. Acquisition and analysis are performed in MetaMorph.
Services
1. Provide training for the operation of a digital imaging microscope for stationary specimens; a confocal microscopy system; and a microscope system for automated real-time imaging and micromanipulation.
2. Provide assistance with the techniques of acquiring and analyzing digital light, fluorescent, and confocal microscopic images, and time lapse, z-series and multiple stage position images using the MetaMorph automated imaging system.
3. Serve as a resource for information on multiple analytical applications of different imaging software currently available at the core imaging facility, and advising on upgrades for future needs and applications.
4. Diagnose and trouble-shooting problems with the imaging microscopes, micromanipulator and gel analysis systems.
5. Integrate data from various imaging systems by designing or implementing software allowing importation of data into MetaMorph for more sophisticated analysis than that available in the software packages that accompany the instrument.
Citation
Please cite the Core Grant for Vision Research - EY08098 in your publications if you have used the services of this module.
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Hybridoma Module
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Purpose
The Hybridoma and Tissue Culture Module accommodates the needs of vision scientists in the Pittsburgh area for biological reagents. It provides extensive expertise in generating hybridomas and production of monoclonal and polyclonal antibodies. Services relating to antibody purification and modification are also offered. In addition, the Module provides explants of various ocular tissues from animal and human donor material, and large-scale preparation of cells is performed. Assistance in generating cells stably expressing recombinant proteins is provided, and the Module maintains a collection of hybridomas and cell lines that are available to all investigators.
The Module interfaces with several of the other Modules. Antibodies produced by the Module are used by investigators in conjunction with the Morphology module and the Electronics and Image Acquisition and Analysis Module for immunolocalization of proteins. Also, the Hybridoma and Tissue Culture Module offers assistance to the Molecular Biology Module in selecting and growing cells stably expressing genetically engineered proteins. Finally, tissue cultures either from primary material or from cell lines will continue to be utilized for analysis by the Gene Expression and Proteomics Module.
Equipment
The facility consists of a 375 square foot tissue culture laboratory located in the OVSRC on the 10th floor of the Eye and Ear Institute. Its central location provides easy access to all participating investigators.
Supplies: The module maintains standard tissue culture supplies including sterile plasticware a variety of media and media components (serum, antibiotics, etc), bioreactor columns for mass production of monoclonal antibodies, RID kits for quantifying antibodies, endotoxin kits, and protein-G and protein-A columns for antibody purification.
Equipment: The Module has two laminar flow hoods and two CO2 incubators that accommodate a roller bottle apparatus and a bioreactor for large-scale production of monoclonal antibodies. The technician has a dedicated desk and a personal computer for maintaining inventories and on-line ordering of supplies. There is also a variety of standard laboratory equipment including centrifuges, a cold box, two inverted microscopes, a dissecting microscope, an autoclave, and water baths for heating tissue culture media.
Services
Preparation of Monoclonal Antibodies from Available Hybridomas
An increasingly broad range of hybridomas are now available commercially or from other investigators, and the trend has been to increasingly use such sources rather than producing the hybridomas in-house. The Module provides help to grow hybridomas in culture, purify the antibodies, test for endotoxin by the Pyrogent® assay (Bio-Whittaker), and quantitate antibodies by a commercially available radial immunodiffusion assay (The Binding Site, Inc.). Large amounts of antibody will be produced by the unique equipment that available to the Module. This includes a large CO2 incubator that accommodates a roller bottle apparatus and a CELLMAX® bioreactor that is capable of simultaneously controlling four hollow fiber bioreactor columns. The column retains monoclonal antibodies in high concentration, while permitting free diffusion of nutrients and allows production of 100-200 mg antibody per month. Smaller amounts of antibody can be produced in cell factories (IBS Integra Biosciences) or roller bottles.
Development of Monoclonal Antibodies
The Module will still retain its capability for producing hybridomas producing specific antibodies, although we expect the usage to decrease. One important reason for offering this service is that it may be desirable to generate a hybridoma to substitute for a heavily used commercially available antibody. For instance, substantial savings have resulted from production of a monoclonal antibody against the hemagglutinin epitope, which is widely used to tag recombinant proteins. The steps in the development of hybridomas secreting monoclonal antibodies of specific interest will include immunization of the mice, fusion of the spleen cells from these mice with myeloma cells, growing the fused cells (hybridomas) in culture, and screening the hybridoma culture supernatants to select the hybridomas secreting specific monoclonal antibodies. This Module has extensive experience in these procedures, and will continue to provide assistance to vision scientists as needed.
Antibody purification and modification. Different researchers require antibodies of varying purities. For some applications, a simple ammonium sulfate fractionation is adequate. However, the Module also has the capacity to purify by protein A or G column chromatography, and the Module director has extensive experience in affinity purification of antibodies, and can assist in setting up such procedures. In vitro applications often require conjugation of monoclonal antibodies to enzymes or fluorochromes. Such applications include: ELISA, immunohistochemical and immunofluorescent staining procedures and Western blots. The Module will provide assistance in conjugation of both monoclonal and polyclonal antibodies to various tags, such as fluorophores and horseradish peroxidase.
Antibody testing and Immunoanalyses
The Module assists researchers in developing polyclonal antibodies. This includes contact with companies that provide immunization and bleeds of animals, and identifying the most cost-effective providers. The Module assays test bleeds for antibody titer. The Module has a considerable expertise in immunological assays, since quite a number of assays have been employed in connection with previous development of monoclonal antibodies and in testing of polyclonal antibodies. These include several variants of ELISA and dot blot assays and radioimmunoassays.
Tissue Culture Techniques
Organ, tissue and cell acquisition. The Module will assist investigators in obtaining cells from animal organs and tissues by acquiring animals or organs and dissecting the required tissues. The Module technician also assists investigators in obtaining human tissues for primary cultures by coordinating the acquisition of pathological and surgical specimens from physicians and the Center for Organ Recovery and Education (a regional organization that serves Western Pennsylvania) and other National Eye Banks.
Primary cell culture and organ culture. The Module carries out protocols for obtaining cells from tissues and organs, including culturing explants and procedures for tissue digestion and separation of desired cell types. For example, several investigators have been growing corneal and conjunctival epithelium and fibroblasts from animal and human tissue explants. After establishing the cell cultures, the Module technician will expand the cells to adequate cell numbers for experiments, perform certain experiments (such as growth curves) or turn the cells over to the investigators.
Culture of cell lines. Maintaining cell lines for investigators is a basic, but immensely useful service that the Module provides.
Large-scale cell culture. The Module assists investigators who require great amounts of cells by preparing large scale cultures in roller bottles and large flasks.
Cryogenic preservation of cells. The Module maintains well-organized stocks of primary and established cell lines so that they are readily available when needed by investigators. Aliquots of cells are stored in a liquid nitrogen tank and maintained by the Module technician.
Mycoplasma and endotoxin testing. Purchasing test kits to assay individual cultures for Mycoplasma and endotoxin contamination is very expensive. However, the cost of such testing is minimized when a single kit is used to test several cell lines. Therefore, the Hybridoma and Tissue Culture Module routinely tests cell lines for Mycoplasma and endotoxin.
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Molecular Biology Module
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Molecular Biology is an essential component of virtually all biological research, particularly in light of the availability of a large volume of sequence information and the ready ability to manipulate it for examination of protein function. Molecular Biology is also a critical component for the follow up to modern genomic and proteomic applications. There has been an explosion in the variety of molecular applications and kits for performing them, and most are constantly changing and developing. As such, molecular biology applies to high throughput assays, manipulation and control of gene expression, mutational analysis of viruses, the host cell or the host animal. It is also a crucial component of most gene knockout and suppression strategies.
The Molecular Biology Module is housed in two rooms (350 sq. ft., and 100 sq. ft.) on the 10th floor of the Eye and Ear Institute.
Equipment
The space includes a separate dark room for core equipment, phosphor imaging and UV gel analysis. Additional equipment required used by the module (ultra and high speed centrifuges, beta counter, etc) are located in a core instrument room, which is located approximately 30 feet from the Molecular Biology Module on the 10th floor. In general, work requests are prioritized on a first come-first served basis for participating NEI-funded investigators; but consideration is also given to small projects requiring immediate attention, investigators seeking pilot data for new vision-related grants, and vision-related research projects that are funded through other NIH institutes.
Major equipment includes hybridization ovens, several vertical and slab electrophoresis apparatuses and the power packs to run them, gel drying apparatus, Stratagene DNA cross-linker used in hybridization studies, UV transilluminator, and digital camera system, cooling and two shaking water baths, refrigerated micro centrifuge, an autoclave, high speed and ultracentrifuges, spectrophotometer and analytical balances. BioRad GS625 Phosphorimager, BioRad EthBr/fluoresce Max Gel and chemiluminescent documentation system, BioRad I cyclker PCR machine, PC computer (networked), and Gene pulser for bacterial transfections. In addition, the module has had access to the electrophoresis equipment of Dr. Kinchington, and has access to P2 tissue culture facilities, high speed and ultra-centrifuges, Speedvac apparatus, -20 and -70 freezers and cold storage, and scintillation counters, all of which are housed in common facilities of the Ophthalmology Research Center and are available for common use on an equally-shared basis.
Services
Preparation and analysis of DNA and RNA
Preparation and screening analysis of phage libraries and generation of subtractive libraries
DNA mutagenesis and expression
DNA:protein, and protein:protein interactions
RNA mapping and accurate quantification
Virus vector design and derivation
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Morphology Module
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The Morphology Module provides technical support for researchers requiring sample preparation for light, confocal, and electron microscopy. These services include fixation of tissues and cultured cells, embedding, section preparation, and staining. The module will also provide facilities and training for individual researchers who prepare and stain their own samples and the module maintains a library of commonly used antibodies and histological reagents available to all core grant users.
Equipment
The lab contains a Leica 2800 Frigocut-E cryostat, Microm HM505E cryostat, a fume hood, light microscope, dissecting scope, refrigerator-freezer and histological technique references. The 10th floor laboratory maintains a library of secondary antibodies labeled with fluorescent tags compatible with filter sets available in the Imaging and Image analysis Module. The Histology Core (2nd floor) provides an Olympus 4060E microscope, a Lancer Vibratome Series 1000, Reichert Jung Autocut Microtome (paraffin & plastics), a Leica-Jung Polycut E fully automated microtome for large sections, a new Leica CM30505 cryostat, a Leica 2145 Microtome, Leica SM 2000R semi automatic sliding microtome, 2 AO Spencer 860 sliding microtomes and 4 AO Spencer 820 rotary microtomes. For electron microscopy there is a JB-4, and 2 LKB ultramicrotomes and an LKB glass knife breaker. For tissue processing a Tissue Tek VIP processor and a Tissue Tek embedding processor are available. A full range of standard histological stains are available. The Core Facility also has access to major equipment such as –80º C freezers, a microwave processor for microwave fixation and antigen recovery methods and a thermal cycler for in situ polymerase chain reaction methods, located on the first floor of the Eye & Ear Institute.
Services
The module technician maintains equipment and reagents in the module laboratory. She works directly with individual researchers advising them on the appropriate fluors for the available microscope filters and secondary antibodies. She carries out fixation, sectioning, and staining of submitted samples. Staining can include standard histological stains, immunofluorescent, or in situ hybridization. The technician will also teach histological procedures to researchers that want to carry out procedures themselves. The technician is responsible for keeping records of services performed and meets on a daily basis with the module director. The module technician coordinates projects with the technicians of the Hybridoma and Tissue culture modules as well as the Molecular Biology Module for use and testing of reagents (such as monoclonal antibodies and labeled probes) common among the modules.
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