The Martin Research Laboratory

This research facility has a 14,000 sq ft of laboratory space consisting of 13 individual laboratories, an iodination room with adjacent isotope and handling area, common instrument rooms for radioactive counters and centrifuges, an acute small animal holding room and a surgical suite, 2 tissue culture areas, a computer and data analysis room and 2 glassware-washing areas. A water distillation system is in service in the laboratory. These areas and the equipment described below are shared by over 20 investigators in the Division of Endocrinology (Departments of Medicine and Pediatrics), the Division of Perinatology (Departments of Pediatrics and Obstetrics) and the General Clinical Research Center (GCRC) Core Laboratories. The faculty and fellows of the Division of Endocrinology ] have full access to the resources of this facility. Laboratory facilities \ include the following:

Morphology Laboratory:The morphology laboratory consists of 350 square feet laboratory space which is equipped for tissue processing, embedding, sectioning, and for routine morphological, biochemical, and molecular biological studies. Equipment include incubators, pH meters, centrifuges, American Optic microscopes, TP 1020 Leica Tissue Processor, AP 280-1 tissue embedding center, paraffin and LKB-ultramicrotomes, power supplies, electrophoresis equipment’s, DNA sequencing equipment’s, shaker baths, fume hoods, freezers and refrigerators, trans-blot apparatus, two thermo-cyclers that are routinely used for polymerase chain reaction (PCR) procedures. A portion of the laboratory is dedicated for in situ hybridization and immunocytochemical studies.

Morphology Core Laboratory: The morphology core is located at the Walter Martin Research Building. It consists of approximately 1000 square feet laboratory space (including Morphology Laboratory described above) and is filly equipped for studies pertinent to testicular and brain pathophysiology using high-resolution light and electron microscopy (see Reproductive Morphology Laboratory described below), morphometry, neuronal and germ cell homeostasis, and in vivo expression of several key genes involved in the regulation of apoptosis. Animal operating rooms, tissue culture, imaging, and dark room facilities are also available. Equipment includes Microscopes (American Optical Microscopes, Nikon Eclipes E800 Photomicroscope, Olympus BH-2 Photomicroscopes), paraffin and ultramicrotomes, a Leica CM 3050 Kryostat, and a Reichert M 1700 microtome. In addition, it has a Nikon Eclipse E800 Photomicroscope, and a Zeiss Axioskop 40 Photomicroscopes attached to an image analysis system for advanced morphometry and semi-quantitative densitometric analysis of immunohistochemical stains This consists of a Zeiss Axiocam color digital camera, Axiovision, Image Pro-Plus software and an advanced computer workstation with graphic accelerators and high storage capacity for image processing.
We have also recently acquired a PALM (stands for positioning and ablation with laser microbeams) microdissection system, which uses Laser Pressure Catapulting (LPC) technology to dissect selected specimens from various sources. This system, which enable fast, easy and reliable non-contact laser micromanipulation and microdissection, consists of a 337 nm pulsed nitrogen laser, a wave length which does not interfere with biological function, coupled into a standard research microscope, RoboStage, CCD color video camera, and fluorescence module, manipulator, and a Zeiss Axiovert 200 microscope.

In addition the Confocal Microscope Facility located in a dedicated laboratory on the second floor of the Martin Research Building houses a Leica TCS SP Upright Confocal Microscope which has three lasers (an argon, a krypton, and a helium-neon lasers) which are connected to the microscope by fiber optics and produce laser lines at 476 (near blue) 488 (blue), 568 (yellow) and 633(far red) nm. The system is equipped with three reflectance/fluorescence photomultiplier/detectors and one for transmitted light (bright field, phase, and darkfield microscopy). This confocal microscope is also capable for Nomarsky optics. The Facility also has a dye sublimation printer for high definition color output.

Aside from the facilities noted above and located in the Martin Research Building, the Morphometric unit utilizes portions of the Histopathology Laboratory, the Transmission Electron Microscopy Laboratory, and the Photography Unit of the Department of Pathology. They comprise approximately 1,600 sq ft. They are all situated on the second floor of the main Hospital Building. This facility is well equipped. The instrumentation includes Hitachi electron microscopes (HU11C and E, and 600); a Zeiss Photomicroscope, a Zeiss Ultraplot, 5 LKB ultratomes, double-headed American Optic and Olympus microscopes, a LabTek II Cryostat, a Reichert sledge M1700 microtome, and 3 fully equipped dark rooms.

Molecular Biology Laboratory: The molecular biology Laboratory is located adjacent to the PI’s Laboratory and consists of approximately 360 sq ft. The equipment includes shakers/incubators for the growth of bacteria and yeast containing recombinant DNA molecules, gel electrophoresis apparatus, DNA sequencing gels and a manifold for dot blot hybridization. In addition, it has two rotating hybridization ovens for Northerns and Southerns, gel transfer apparatus, photographic equipment for autoradiography and A BioRad Fluro-S-Multimager for visualization and analysis of autoradiograms, chemiluminescent and fluorescent gels. Protein electrophoresis equipment includes several BioRad mini-gel apparatuses, and a Hoeffer Western Blot Transfer apparatus. Recently acquired equipment include an Affymetrix GeneChip Analysis Suite and two ABI PRISM 7000 real time PCR machines in a dedicated laboratory located on the second floor of the Walter P Martin research building. The GeneChip Analysis Suite consists a Gene Chip hybridization oven, fluidics station and a GeneArray TM scanner. Software includes state-of-the art software for gene expression studies using high-density oligonucleotide arrays including a LIMS Manager, data mining tools, and Genespring software for Cluster analysis of gene expression data. Recently, the institution further acquired two ABI PRISM 7000 real time PCR machines.

Gas-Liquid Chromatograph/Mass Spectrometer Facility: The Shared Instrumentation Program of the Division of Research Resources awarded the Harbor-UCLA LABioMed a Hewlett-Packard, Model 5985B gas-liquid chromatograph/mass spectrometer (GCMS). This facility receives support from the GCRC, the Clinical Nutrition Research Unit, the Research Committee of the Harbor-UCLA Medical Center, and from individual investigators who use it. It is located in the Martin Research Building. The system is capable of continuous scanning with a specified mass range or simultaneous ion monitoring of a few specified masses. Very small amounts of material (e.g., 100 ul plasma) are needed to quantitate nmol or pmol amounts.

Semen Analysis Laboratory: The Semen Analysis Laboratory consists of 200 square feet space, and is equipped with an Olympus BH-2 photomicroscope both for light and fluorescence microscopy and an Olympus BH-2 microscope attached with “Cell Traks System” and a Hamilton-Thorn machine for computer added semen analysis. The Semen analysis laboratory is located next to the clinical examination rooms on level 2 of the Martin Research Building.

Urology Research Unit: The Urology/Andrology Research Laboratories consist of 3,800 square feet space, and comprises tissue culture, biochemistry and molecular biology wet laboratories, small animal operating rooms and dark room facilities. Drs. Cadavid and Rajfer and their staff of postdoctoral fellows, research technicians, and visiting scientists are located here. Drs. Cadavid and Rajfer are members of the Harbor-UCLA Reproductive Endocrinology group and participate in studies on reproductive dysfunction, infertility, and contraceptive development.

Shared Equipment: Counting equipment includes 2 Searle autogamma counters, a small hand held (non-automated) gamma counter for iodination screening and 2 Micromedic multichannel 600 tube programmable automatic gamma counters with an attached IBM computer for data analysis. In addition, there is 1 Beckman automatic beta counter and 1 Searle automatic beta counter.

Centrifuges available for common use include three low speed refrigerated centrifuges, 2 high speed Sorvall refrigerated centrifuges, and two Beckman preparative ultracentrifuges. The individual labs are also equipped with desktop non-refrigerated centrifuges which are used for both tissue culture cell separation and other preparatory steps.

Automatic radioimmunoassay equipment includes many desktop automated and semi-automated pipettors in individual laboratories.

HPLC equipment includes 1 IBM ternary gradient HPLC, 1 Beckman System Gold and 1 Waters HPLC with autosampler dedicated to catecholamine analysis.

Spectrophotometers and fluorometers are in use include a Beckman DU 640 spectrophotometer, an ABA chemistry analyzer and a Perkin/Elmer fluorometer, also capable of measuring bioluminescence and phosphorescence. There is also an LKB Wallace 1130 Arcus time resolved fluorometer with platewasher.

Molecular biology equipment includes 1 Protean II electrophoretic apparatus (BioRad), 3 Ames electrophoretic apparatuses, Hoeffer Western Blot transfer apparatus, LKB electrofocusing equipment, temperature regulate circular water bath, photographic equipment for autoradiography and 2 manifolds for dot blot hybridization. A video densitometer is available for part-time use. DNA sequencing equipment and a Perkin Elmer/Cetis DNA thermocycler for polymerase chain reaction are available.

Tissue culture equipment consists of 3 laminar flow hoods, 3 Co2 humidified atmosphere incubators, an autoclave, two inverted photomicroscopes with light, dark field and phase contrast capabilities, a liquid nitrogen cell bank and microfuges for cell separation.

Recently we have also acquired a complete Gene Chip Analysis Suite, including GeneArray TM Scanner, LIMS Manager, data mining tool, and state-of-the art software for gene expression studies using high-density oligonucleotide arrays (Affymetrix Gene Chip Technology, Santa Clara, CA).

Tissue Culture Facility: The shared cell culture facility of WALTER P MARTIN Research Building is an 800 sq ft three-room compartment that is connected through safety doors. Each room is assigned to perform different cell culturing tasks: The main room (400 sq ft) contains the two recently certified laminar flow biological safety cabinets and one EdgeGard hood with continuous horizontal air flow. In this room there are 6 Forma Scientific CO2 and temperature controlled Water-Jacket tissue culture incubators for cell culturing. The two phase-contrast inverted microscopes are used to study cell cultures, potential contamination, cell growth and confluence of the cultures. The middle room (300 sq ft) has two Locator Cryo biological storage systems, cooled with liquid nitrogen and this is where the CO2 tanks are stored to supply the tissue culture incubators. The smallest room (200 sq ft) is designated for specific tissue culturing tasks including animal tissue or infectious material and is equipped with one EdgeGard hood with continuous horizontal air flow, three tissue culture incubators and a phase-contrast inverted microscope. This culture facility is regularly serviced by the Biological and Chemical Safety Office of our Institution and equipped with chemical and biological safety containers for the disposal of cell remains.

Flow Cytometry Core Facility: Harbor-UCLA Research and Education Institute supports a modern Flow Cytometry Core Facility featuring a Becton-Dickinson FACSCaliburä cytometer configured with in-line fluorescence-activated cell sorting. This system has several features that are advantageous to multiple applications in molecular and cell biology. The FACSCaliburä is equipped with two distinct laser sources (argon and helium-neon), and an array of detectors (FSC, SSC, and FL1-FL4), enabling investigators to perform simultaneous four-color analysis. This feature is crucial for performing multi-color analyses examining three or more coincident assessments of cellular function. Furthermore, the dual-laser configuration used in the FACSCaliburä achieves a fluorescence sensitivity equivalent to a detection limit of 750 FITC molecules per particle, and operates with a standard resolution of 3% variance. This degree of sensitivity and resolution is essential in detecting rare subpopulations of cells, or when using dyes that emit fluorescence wavelengths differing by 50 nm or less. Additionally, the FACSCaliburä cytometer is equipped with an in-line cell sorter, allowing investigators to analyze and isolate particular cells based on analysis, gating, and/or fluorescence activation. The cell sorting device available for the FACSCaliburä is capable of sorting four distinct fluorochrome labels with a sorting efficiency of 95% purity, and allows for single cell, exclusion, and recovery modes of collection. The FACSCaliburä flow cytometer interfaces with computer-based analysis and sorting programming, and data management station (FACStationä). The configuration provides user-friendly, menu driven software for instrument set up, tuning, and calibration, as well as for analysis, data collection and management. The FACStationä software also performs data analysis using plotting, gating, and statistical modes, and generates high quality graphic reports suitable for publication.