Director
Juliet A. Greenwood, Ph.D.
julie.greenwood@oregonstate.edu
541-737-4997

Developing Technologies

  • NSF-MRI for two-photon confocal twin system submitted in collaboration with CGRB
  • NIH S10 shared instrument grant for a micro-CT
  • Imaging of superoxide in living cells
  • Imaging of fluorescent proteins with unnatural amino acids

Instrumentation/Services

Publications

Cell imaging Instrumentation

 

Molecular Devices High-Content Imager

The ImageXpress Micro widefield high content screening system provides a fully automated solution to capture high-quality images of fixed- or live-cell assays.  While the high throughput capability of this system is best achieved using samples in multi-well optical plate format, the system can also accommodate samples, live or fixed, on standard glass microscope slides.  Five objective lenses (1X, 2X, 10X, 20X, 40X) are available (10X and 20X lenses support phase contrast); images may be captured under brightfield (halogen lamp) and/or fluorescence (mercury lamp) illumination.  Optical filter sets provide for excitation (350-400 nm, 450-490 nm, 525-560 nm) and emission (415-480 nm, 500-540 nm, 570-620 nm) ideal for tracking fluorescent dyes such as DAPI, FITC, and TRITC, respectively.  The sample chamber can be temperature-controlled (heating only) and an optional sample plate manifold makes it possible to perform live cell work under an air/CO2 atmosphere mix.  A dedicated PC workstation equipped with MetaXpress software (recently upgraded to v4.4.4.43) is available for collecting and analyzing images; images are stored (and automatically backed up daily) in a database residing on a remote server.  A second PC workstation equipped with MetaXpress (v3.1) is available for post-acquisition image analysis.

MetaXpress software is modular in nature; the current package installed includes the following suite of applications:

  • Angiogenesis Tube Formation – for tube formation of new blood vessels, both in health and disease
  • Multi Wavelength Cell Scoring – for counting and logging measurements of cells in multiple wavelength experiments
  • Multi Wavelength Translocation – for simultaneous measurement and scoring of up to six translocating probes, ideal for signal transduction and pathway profiling studies
  • Neurite Outgrowth – for studying extension of axonal processes from the cell body, which can be a natural part of early development but is also implicated in a broad range of CNS disorders or injuries
  • Transfluor – designed to facilitate analysis of receptor internalization

 

 


Zeiss PALM MicroBeam IV Laser Capture System

Work in the Laser Capture Microdissection (LCM) facility revolves around a Zeiss AxioObserver Z1 inverted microscope equipped with objective lenses (5X, 10X, 20X, 40X) optimized for brightfield, fluorescence, and laser capture .  A MicroBeam UV laser (355 nm) provides for microdissection with 0.6 micron resolution.  A joystick- and/or keyboard-controlled, robotic stage accommodates samples mounted on standard microscope slides and/or in tissue culture dishes; a robotic collector mounted above the stage provides for automated sample collection directly into microfuge or PCR tubes.  Typical brightfield illumination is provided by a halogen lamp; a suite of Colibri LED lamps provides for fluorescence at 365 nm (blue), 470 nm (green), 490 nm (red), and 565 nm (far red) wavelengths.  Two digital cameras are available: The AxioCam 1Cc1 color camera (1.4 megapixels) is used for brightfield imaging while the AxioCam MRm monochrome camera (1.4 megapixels) is best for fluorescence imaging.  The entire microscope assembly is contained within a transparent incubator that provides a completely enclosed, temperature-controlled environment for live cell work.  Images and laser capture are monitored on two displays – one conventional widescreen computer monitor and an additional, touchscreen, monitor that is stylus-equipped and can be used to designate laser capture targets.  All components are integrated and controlled using a PC workstation running the Windows 7 operating system.  Two software packages, PALMRobo v4.5 and AxioVision v4.8, provide for automated laser capture and image analysis, respectively.

Recent publications: 

Rhoads T.W., Williams J.R., Lopez N.I., Morré J.T., Bradford C.S., Beckman J.S. (2012) Using Theoretical Protein Isotopic Distributions to Parse Small-Mass-Difference Post-Translational Modifications via Mass Spectrometry.  J Am Soc Mass Spectrom. 2012 Dec 18. [Epub ahead of print]  PMID: 23247967.

Rong Wang, Wan-Mohaiza Dashwood, Hui Nian, Christiane V. Lohr, Kay A. Fischer, Naoto Tsuchiya, Hitoshi Nakagama, Hassan Ashktorab, and Roderick H. Dashwood (2010) NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).  Int. J. Cancer 128: 2581-90.  PMID: 20715105.


For more information:  http://corelabs.cgrb.oregonstate.edu/lcm


Seahorse Biosciences Extracellular Flux Analyzer

This instrument provides an intermediate-throughput platform for rapid and sensitive identification of environmental chemicals altering cellular bioenergetics by measuring the two major energy producing pathways of the cell, mitochondrial respiration and glycolysis, in real-time.  The Analyzer provides a 24-well format to simultaneously perform time-resolved measurements of the oxygen consumption rate and extracellular acidification rate in a single population of cells over a period of minutes, hours, or even days.  The label-free assay system allows reuse of the cells following measurement.  Instrument software guides the user through the experimental design and provides a summary report on completion.  In addition, the system is accompanied by the XF Prep Station to prepare culture plates by providing fast and thorough washing of the cells for consistent assay conditions.

Seahorse Biosciences site for videos, webinars, and publications: http://www.seahorsebio.com/

New User Packet: link in progress

Workshop presentations: link in progress

 


xCELLigence Real-Time Cell Monitoring System

The critical and unique feature is that the xCELLigence System detects cell responses throughout an experiment, enabling discovery not possible with endpoint assays.  The xCelligence System provides improved, real-time, label free and non-invasive analyses of key cellular events with an impedance based cell sensing measurement system. The System consists of an analyzer that can fit on one half of one shelf of a standard cell culture incubator.  The analyzer has slots for three specialized plates containing 16 wells each.  Therefore, up to 48 conditions or treatments can be monitored at one time.  Applications include: Cell invasion and migration assays, Compound- and cell-mediated cytotoxicity, Cell adhesion and cell spreading, Cell proliferation and cell differentiation, Receptor-mediated signaling, Virus-mediated cytopathogenicity.

Recent publications:

Wang, R., Lohr, C.V., Fischer, K.A., Dashwood, W.M., Greenwood, J.A., Ho, E., Williams, D.E., Ashktorab, H., Dashwood, M.R., and Dashwood, R.H. (2012) Epigenetic inactivation of endothelin-2 and ET-3 in colon cancer.  Int. J. Cancer, 132, 1004-12.  PMID: 22865632

O’Donnell, E.F., Kopparapu, P.R., Koch, D.C., Jang, H.S., Phillips, J.L., Tanguay, R.L., Kerkvliet, N.I., Kolluri, S.K.  (2012) The Aryl Hydrocarbon Receptor Mediates Leflunomide-Induced Growth Inhibition of Melanoma Cells. PLoS ONE 7(7): e40926.  PMID: 22815870

Acea Biosciences website:  http://www.aceabio.com/product_info.aspx?id=184

User protocols: link in progress 

 


Zeiss lSM 510 META Confocal Microscope

The CIAFC operates a Zeiss LSM 510 META Confocal Microscope, which contains four lasers providing seven laser lines for imaging a wide variety of fluorescent probes. In addition, the META detector can separate highly overlapping spectra. An array of objectives is available to accommodate diverse sample types from cultured cells to animal or human tissue. The system contains a physiology incubation chamber for imaging live cells. The manufacturer designed the confocal system’s operating software for specialized applications such as fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET) in fixed and live cells. The Confocal Microscope resides in ALS2070, a low-vibration room near the cell culture rooms and Dr. Greenwood’s laboratory.

Recent publications:

Lal, S., La Du, Jane, Tanguay, R.L., and Greenwood, J. A. (2012) Calpain 2 is required for the invasion of glioblastoma cells in the zebrafish brain microenvironment.  J. Neurosci. Res., 90, 769-81.  PMID: 22183788

Campbell, A.L., Shih, H.P., Xu, J., Gross, M.K., Kioussi, C. (2012) Regulation of motility of myogenic cells in filling limb muscle anlagen by Pitx2.  PLoS One 7, 335822.  PMID 22558231

For more information: http://corelabs.cgrb.oregonstate.edu/confocal

 


Zeiss Axiovert 100S Widefield Microscope

In addition, the facility is equipped with a Zeiss Axiovert 100S widefield microscope for examining fixed or live cells via fluorescence, phase contrast, differential interference contrast, and interference reflection microscopy. The microscopy system contains a Photometrics CoolSNAP HQ CCD camera, a high-resolution digital camera that can image in the near-infrared. MetaMorph Imaging Software controls image acquisition and provides extensive analysis functions.

 


Eppendorf Semi-Automatic Microinjection System

The facility contains an Eppendorf semi-automatic microinjection system for experiments that require rapid injection of DNA, protein, and other molecules into adherent cells.

 


Cell Culture Rooms

The facility includes four cell culture rooms equipped with laminar-flow hoods, incubators, water baths, tabletop centrifuges, phase and fluorescence microscopes, refrigerators/freezers (4oC/-20oC/-85oC), and large-scale roller bottle/suspension culture equipment. One of the cell culture rooms and hood is approved for Biosafety Level 2 work. The facility also contains a KODAK Image Station 440CF, SpectraCount microplate reader, BioRad SmartSpec 3000, Beckman J2-21 centrifuge, Beckman J-6B centrifuge, autoclave, microfuge, chemical fume hoods, water purification system, microwave and baking oven, pan and analytical balances, pH meter, and sonicator. The core maintains cryogenic freezers for long-term storage of cells in liquid nitrogen. CIAFC staff maintains a computerized inventory of all cells to facilitate organization and retrieval of cell lines.

 


Publications

Lal, S., La Du, Jane, Tanguay, R.L., and Greenwood, J. A. (2012) Calpain 2 is required for the invasion of glioblastoma cells in the zebrafish brain microenvironment.  J. Neurosci. Res., 90, 769-81.  PMID: 22183788

Wang, R., Lohr, C.V., Fischer, K.A., Dashwood, W.M., Greenwood, J.A., Ho, E., Williams, D.E., Ashktorab, H., Dashwood, M.R., and Dashwood, R.H. (2012) Epigenetic inactivation of endothelin-2 and ET-3 in colon cancer.  Int. J. Cancer, 132, 1004-12.  PMID: 22865632

Smith, M., Boenzli, M., Hindagolla, V., Ding, J., Miller, J., Hutchison, J., Greenwood, J.A., Abeliovich, H., and Bakalinsky, A. (2012) Identification of gold nanoparticle-resistant mutants of Saccharomyces cerevisiae suggests a role for respiratory metabolism in mediating toxicity.  Appl. Environ. Microbiol., 79, 728-33.  PMID: 23144132

Campbell, A.L., Shih, H.P., Xu, J., Gross, M.K., Kioussi, C. (2012) Regulation of motility of myogenic cells in filling limb muscle anlagen by Pitx2.  PLoS One 7, 335822.  PMID 22558231

Rhoads T.W., Williams J.R., Lopez N.I., Morré J.T., Bradford C.S., Beckman J.S. (2012) Using Theoretical Protein Isotopic Distributions to Parse Small-Mass-Difference Post-Translational Modifications via Mass Spectrometry.  J Am Soc Mass Spectrom. 2012 Dec 18. [Epub ahead of print]  PMID: 23247967

O’Donnell, E.F., Kopparapu, P.R., Koch, D.C., Jang, H.S., Phillips, J.L., Tanguay, R.L., Kerkvliet, N.I., Kolluri, S.K.  (2012) The Aryl Hydrocarbon Receptor Mediates Leflunomide-Induced Growth Inhibition of Melanoma Cells. PLoS ONE 7(7): e40926.  PMID: 22815870

Rohlman, D., Pham, D., Yu, Z., Steppan, L., and Kerkvliet, N. (2012). Aryl hydrocarbon receptor-mediated perturbations in gene expression during early stages of CD4+ T-cell differentiation. Frontiers in Immunology 3, 223.  PMID: 22888330 

Corsini, E., Oukka, M., Pieters, R., Kerkvliet, N.I., Ponce, R., Germolec, D.R. (2011) Alterations in regulatory T-cells: rediscovered pathways in immune-toxicology. J. Immunotoxicol. 8, 251-7. PMID: 21848365

Kerkvliet, N.I. (2012) TCDD: An environmental immunotoxicant reveals a novel pathway of immunoregulation – a 30-year odyssey.  Toxicol. Pathol. 40, 138-42. PMID: 22089840

Jason L. Seitchik, Jennifer C. Peeler, Michael T. Taylor, Melissa L. Blackman, Timothy W. Rhoads, Richard B. Cooley, Christian Refakis, Joseph M. Fox, and Ryan A. Mehl. Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific in Vivo Bioorthogonal Ligation with trans-Cyclooctenes. J. Am. Chem. Soc. 2012, 134(6), 2898-901. PMID: 22283158

Ryan A. Mehl. Engineered Unnatural Animals: Tools for Multicellular Biochemistry. ChemBioChem. 2012 13(2), 186-8.  PMID: 22351958

Shorey, L.E., Castro, D.J., Baird, W.M., Siddens, L.K., Löhr, C.V., Matzke, M.M., Waters, K.M., Corley, R.A., Williams, D.E. (2012) Transplacental carcinogenesis with dibenzo[def,p]chrysene (DBC): timing of maternal exposures determines target tissue response in offspring. Cancer Lett 317, 49-55.  PMID: 22085489

Siddens, L.K., Larkin, A., Krueger, S.K., Bradfield, C.A., Waters, K.M., Tilton, S.C., Pereira, C.B., Löhr, C.V., Arlt, V.M., Phillips, D.H., Williams, D.E., Baird, W.M. (2012) Polycyclic aromatic hydrocarbons as skin carcinogens: comparison of benzo[a]pyrene, dibenzo[def,p]chrysene and three environmental mixtures in the FVB/N mouse. Toxicol Appl Pharmacol 264, 377-386.  PMID: 22935520

Corsini, E., Oukka, M., Pieters, R., Kerkvliet, N.I., Ponce, R., Germolec, D.R. (2011) Alterations in regulatory T-cells: rediscovered pathways in immune-toxicology. J. Immunotoxicol. 8, 251-7.  PMID: 21848365

Ma, L., Greenwood, J.A., and Schachner, M. (2011) CRP1, a protein localized in filopodia of growth cones, is involved in dendritic growth.  J. Neurosci. 31, 16781-91. PMID: 22090504

McDevitt, R.I., Ruaux, C.G., and Baltzer, W.I.  (2011) Influence of transfusion technique on survival of autologous red blood cells in the dog.  Journal of Veterinary Emergency and Critical Care, 21:209-216. PMID: 21631706

Saili K.S., Corvi M.M., Weber D.N., Patel A.U., Das S.R., Przybyla J., Anderson K.A., and Tanguay R.L. (2012) Neurodevelopmental low-dose bisphenol A exposure leads to early life-stage hyperactivity and learning deficits in adult zebrafish.  Toxicology 291(1-3):83-92. PMID: 22108044; PubMed Central PMCID: PMC3245816.

Wang, R., Dashwood, W.-M., Nian, H., Löhr, C.V., Fisher, K.A., Tsuchiya, N., Nakagama, H., Ashktorab, H., and Dashwood, R.H. (2011) NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Int J Cancer. 128(11), 2581–2590. PMCID: PMC3262595

O'Donnell EF, Saili KS, Koch DC, Kopparapu PR, Farrer D, Bisson WH, Mathew LK, Sengupta S, Kerkvliet NI, Tanguay RL, Kolluri SK. (2010) The Anti-Inflammatory Drug Leflunomide Is an Agonist of the Aryl Hydrocarbon Receptor. PLoS One. 2010; 5(10). pii: e13128.PMID: 20957046

Larsen, C.A. and Dashwood, R.H. (2010) Epigallocatechin-3-gallate inhibits Met signaling, proliferation, and invasiveness in human colon cancer cells.  Arch. Biochem. Biophys. 501, 52-7.  PMID: 20361925

Rong Wang, Wan-Mohaiza Dashwood, Hui Nian, Christiane V. Lohr, Kay A. Fischer, Naoto Tsuchiya, Hitoshi Nakagama, Hassan Ashktorab, and Roderick H. Dashwood (2010) NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).  Int. J. Cancer, In press.

Jang, H.S., Lal, S., and Greenwood, J.A. (2010) Calpain 2 is required for glioblastoma cell invasion: regulation of matrix metalloproteinase 2.  Neurochem. Res. 35, 1796-1804.

MARSHALL, N.B.  and  KERKVLIET, N.I.  (2010). Dioxin and immune regulation. Emerging  role of aryl hydrocarbon receptor in the generation of regulatory T cells. In: The Year in Immunology II, Noel R. Rose, Ed.  Ann. NY Acad. Sci. 1183: 25 – 37, 2010.

Löhr CV, DeBess EE, Baker RJ, Hiett SL, Hoffman KA, Murdoch VJ, Fischer KA, Mulrooney DM, Selman RL, Hammill-Black WM. Pathology and viral antigen distribution of lethal pneumonia in domestic cats due to pandemic (H1N1) 2009 influenza A virus. Vet Pathol. 2010 May;47(3):378-86.

Hsu A, Bruno RS, Löhr CV, Taylor AW, Dashwood RH, Bray TM, Ho E. Dietary soy and tea mitigate chronic inflammation and prostate cancer via NFkappaB pathway in the Noble rat model. J Nutr Biochem. 2010 Aug 27.

FUNATAKE, C.J., AO, K., SUZUKI, T, MURAI, H., YAMAMOTO, M., FUJII-KURIYAMA, Y., KERKVLIET, N.I. and NOHARA, K. (2009) Expression of Constitutively Active Aryl Hydrocarbon Receptor in T-Cells Only Enhances the Down-regulation of CD62L, But Does Not Alter Expression of CD25 or Suppress the Allogeneic CTL Response. J Immunotoxicol. 6: 194-203.

Bajaj, G., Zhang, Y., Schimerlik, M.I., Hau, A.M., Yang, J., Filtz, T.M., Kioussi, C. and Ishmael, J.E. (2009).  NMDA receptor subunits are non-myosin targets of myosin regulatory light chain. J. Biol. Chem 284: 1252-1266 PMC2613636

Korakod Chimploy, G. Dario Dıaz, Qingjie Li, Orianna Carter, Wan-Mohaiza Dashwood, Christopher K. Mathews, David E. Williams, George S. Bailey, and Roderick H. Dashwood (2009) E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin. Int. J. Cancer: 125, 2086–2094.

KERKVLIET, N.I., STEPPAN, L B., VORACHEK, W., ODA, S., FARRER, D., WONG, C.P., PHAM, D. and MOURICH, D.V. (2009) Activation of the Aryl Hydrocarbon Receptor by TCDD Prevents the Development of Diabetes in NOD Mice and Increases Foxp3+ T Cells in Pancreatic Lymph Nodes. Immunotherapy 1: 539-547.

DJ Castro, CV Löhr, KA Fischer, K Waters, BJ Webb-Robertson, RH Dashwood, GS Bailey, DE Williams. Identifying efficacious approaches to chemoprevention with chlorophyllin, purified chlorophylls and freeze-dried spinach in a mouse model of transplacental carcinogenesis. Carcinogenesis (2009) 30:315-320.

Q Li, CV Löhr, RH Dashwood. Activator protein 2alpha suppresses intestinal tumorigenesis in the Apcmin mouse. Cancer Letters (2009) 283:36-42.

Nian H, Bisson WH, Dashwood WM, Pinto JT, Dashwood RH. Carcinogenesis. 2009 Aug;30(8):1416-23. Epub 2009 Jun 15.  Alpha-keto acid metabolites of organoselenium compounds inhibit histone deacetylase activity in human colon cancer cells. 

Schaefer D, Bildfell RJ, Löhr CV: Placental insufficiency as possible cause of llama and alpaca abortions. Veterinary Pathology- accepted.

Yu Z, Chung WG, Sloat BR, Löhr CV, Weiss R, Rodriguez BL, Li X, Cui Z. The extent of the uptake of plasmid into the skin determines the immune responses induced by a DNA vaccine applied topically onto the skin. Journal of Pharmacy and Pharmacology. In print.

Jang, H.S. and Greenwood, J.A. (2009) Gylcine-rich region regulates cysteine-rich protein 1 binding to actin cytoskeleton.  Biochem. Biophys. Res. Commun. 380, 484-488.