Cell Biology and Flow Cytometry Facility

The 2,600 sq. ft. DBMR Cell Biology and Flow Cytometry Facility is located near the Link Engineering Building. The facility houses four laboratories whose research focus includes: 1) mechanism of action studies of marine-derived compounds in the areas of immunology and tumor biology 2) design and development of novel screening assays for the high throughput laboratory and 3) studies on the taxonomy and cell culture of marine invertebrates. Each laboratory of the facility is fully equipped to study the cellular and molecular aspects of living cells of both mammalian and invertebrate species. An integral part of these studies relies on the use of flow cytometric analysis which is provided by a separate flow cytometry laboratory located within the building. In addition, the facility provides a small vivarium for experimental in vivo studies and a separate laboratory for radioisotope usage. HBOI is licensed under the State of Florida (Site License #1034-1) to handle most commonly utilized radioisotopes.

Equipment & Facilites:   • Biological Safety Cabinets (laminar flow hoods for each laboratory)   • Incubators   • LabLine EnvironShaker Incubator   • Olympus Inverted (2) and Standard Microscopes (2)   • Olympus Research Fluorescence Microscope with Photomicrography Attachments   • Refrigerated Centrifuge (2)   • Freezers and Refrigerators   • REVCO -80 Freezer   • Shannon Cyto-Centrifuge   • BioTech Model EL-311 Mictotiter 96-Well Plate Reader   • Ph.D. Cell Harvester   • Beckman LS3801 Liquid Scintillation Counter   • Perkin Elmer Fluorescence Spectrophotometer   • Coulter EPICS Elite Flow Cytometer (3 laser configuration, gated amplifier, autoclone)

Other equipment includes pH meters, balances, microfuges, osmometer, water purification system, pull-down isolation and microisolator cage units (in vivarium), cage washer and autoclave. Each laboratory also has available P.C.'s, which are configured with Windows for Workgroups and are connected to a campus-wide network that provides access to the Internet.

Mechanistic studies on pure compounds and design of new screening assays routinely utilize column chromatographic, protein and DNA electrophoretic equipment located in the facility.

Screening Laboratory

The Screening and Biochemistry groups occupy a 1,500 sq.ft. suite of laboratories within the Link Engineering Building. The laboratory is able to handle a broad range of screening projects derived from both in-house research and from external collaborations. Current primary screen throughput rates are from 500-1000 samples per run of an assay. The screening capacity of the laboratory is in excess of 10,000 assay points per week. The equipment allows the determination of colorimetric, radioactive, luminescent and fluorescent endpoints. The HBOI site license allows the use of all routinely-used radioisotopes.

Equipment & Facilities:   • Two Tecan automated liquid handlers (Models 8051 and Megaflex)   • SLT 96-well plate spectrophotometer   • Wallac Betaplate scintillation counter   • Perkin Elmer fluorescence spectrophotometer equipped for 96-well plate measurements   • Packard TopCount microplate scintillation counter (to be added this year)   • Beckman LS3801 liquid scintillation counter   • TomTek 96-well cell harvester   • Centrifuges (Beckman J2-HS, Beckman L7 ultracentrifuge, Sorvall RT6000B)   • Incubators (3 cabinet incubators including one CO2 incubator; 1 Gallenkamp orbital incubator equipped for sub-ambient operation)   • Freezers (-20C (Lab-Line solvent freezer), -80C and -140C (Revco))   • Biological safety cabinets (2 6ft Baker; 1 6ft NuAire; 1 4ft BBL)

Other equipment includes pH meters, balances, microfuges, equipment for cell disruption and a Barnstead Nanopure water purification system. An autoclave is located close to the laboratory.

The fully equipped biochemistry laboratory is able to perform gel electrophoresis of proteins and nucleic acids, PCR, and column chromatography. A Pharmacia FPLC system is routinely used in protein purification.

Fermentation Laboratory

The goals of the Fermentation Laboratory are to isolate microorganisms such as bacteria and fungi from the marine environment and to ferment these microorganisms to provide extracts for drug discovery programs. Isolation sites have included the Caribbean, the Gulf of Mexico, the Eastern Atlantic and the Pacific Oceans. The emphasis is on deep water sources collected by submersible at depths to 3,000 feet. Microorganism isolation is started on shipboard using freshly collected material. The current collection contains approximately 8,000 strains. New isolates are added to the collection at a rate of 1,500 per year. The majority (62%) of the strains were isolated from marine macroorganisms with the balance derived from other marine sources such as sediments.

Equipment & Facilities: • Consolidated autoclave   • Barnstead water purification system   • Beckman J2-21 centrifuge and rotors   • Baker and Labline biohoods   • Two chemical fume hoods   • Olympus microscope (phase contrast, epiflourescence, and photomicrograph capabilities)   • Two Reichert dissecting microscopes   • Water baths   • Mettler balances (top loading and analytical)   • Five incubators (Equatherm and Labline)   • Two freeze dryers (Virtis 15 SRC-X and Virtis Freezemobile 24)   • Brinkman rotovap system   • Ultra-low freezer (Revco ULT-2090-5 Elite Chest Freezer)   • Four Labline orbital shakers   • New Brunswick incubating orbital shaker   • Jouan RC1010 centrifuge evaporator   • Spectra 96-well plate reader   • Miscellaneous: refrigerators, freezers, hot plates, two Zeos 486 computers

The isolation and cultivation of the microogranisms is performed in a dedicated microbiology facility, a detached building of 1300 sq. ft. floor space.

Natural Products Chemistry Laboratories

Each chemist works on two to four projects concurrently. Our goal is to identify the class of bioactive compound within one month in order to make a decision as to whether the class of compound is useful as a drug lead prior to complete characterization of the compound. The average time for completion of bioassay guided isolation and structure elucidation is 3 months. Typically, twenty to thirty compounds are characterized annually.

Equipment & Facilities: • Four 1000 sq. ft. chemistry labs   • One 1000 sq. ft. extraction Laboratory   • One 1500 sq. ft. instrumentation laboratory   • Seven tertiary gradient HPLC systems equipped with variable wavelength UV-VIS and refractive index detectors (one fluorescence detector and one diode array detector also available)   • Ito Multilayer Planetary Coil Countercurrent Chromatography Unit   • Chromatotron Preparative TLC system   • Preparative scale HPLC system   • Four MPLC systems   • Bruker AMX 500- Equipped for Normal and Inverse detection; Multiple Probe Heads (5 mm Dual Probe, 5 mm   • Broadband Inverse Probe, 10 mm Broadband Dual Probe); Capable of running most common 1-D, 2-D and 3-D experiments   • Bruker AM-360 with Aspect 3000 Computer; Equipped for Normal and Inverse detection, Multiple Probe Heads (5 mm   • Dual Probe, 5 mm Broadband Inverse Probe, 10 mm Broadband Dual Probe); Capable of running most 1-D and 2-D experiments.   • Iris Indigo Work Station: Tripos, Sybil with Triad Software for Modeling and NMR processing   • Multiple PCs (486 and Pentium) running Isis Draw, Isis Base, PC Model, MS Access, MS Excel, MS Word, MS Powerpoint, Visio   • Databases: Proprietary Marine Natural Products Database; Chapman and Hall Natural Products Database; Faulkner Marine Database; Munro Marine Database; CAS on-line   • Midac FT-IR   • Perkin Elmer Lambda 3B Uv-vis spectrophotometer   • Polarimeter (Jasco DIP-360).   • Miscellaneous: Ten rotatory evaporators, Two Jouan evaporators, Two Vapotec Evaporators, sonication baths, drying ovens, HPLC and MPLC columns (RP-18, RP-8, RP-4, RP-2, Phenyl-CN, Diol, Amino, Silica, etc.)   • Standard Stationary phases: RP-18, RP-8, RP-4, Silica, Sephadex LH-20 and G- series, BioGel P-2, BioRad Bio Bead series, HP-Diaion-; TSK-gel; BioRad MSZ-1, XAD resins etc..); miscellaneous glassware for isolation and synthetic chemistry.

Molecular Genetics Laboratory

The 200+ sq. ft. Molecular Genetics laboratory is equipped to perform many routine molecular experiments. Current projects include a) application of genetic fingerprinting and DNA sequence analysis for identification of marine invertebrate cell types grown in vitro; b) cloning of species-specific cell growth factors; c) molecular evolutionary analyses of secondary metabolite biosynthetic pathways; d) sponge-microbe symbioses and e) marine invertebrate molecular systematics and population genetics.

Equipment & Facilities: Laboratory facilities include programmable thermalcyclers for PCR experiments (an MJ Research Peltier PTC-1160, a Perkin-Elmer 480), agarose gel apparatuses, polyacrylamide gel systems, Stratagene Eagle Eye II photosystem for electronic gel documentation, GeneQaunt and UV spectrophotometer for nucleic acids quantification, microcentrifuges, and Beckman G5-15R and Sorvall RT6000B refrigerated table top centrifuges. Facilities are also available for radioactive detection methods. DNA sequencing gels are run off-campus, but onsite sequence analysis software includes PAUP* 4.0, PHYLIP, CLUSTAL W, which are run on either Macintosh PowerPCs (603e and G3 coprocessors) and Pentium-based IBM compatible PCs. Full internet access to other analytical software (GCG) is also available.