29 articles published in JoVE
1Laboratory of Cell and Molecular Biology, University of Wisconsin – Madison, 2Department of Genetics, University of Wisconsin – Madison
To complement commonly used methods to study TRPV4’s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.
Published December 31, 2013. Keywords: Basic Protocol, Eukaryota, Archaea, Bacteria, Life Sciences (General), Mechanosensation, Ion channels, Lipids, patch clamp, Xenopus Oocytes, yeast, luminometry, force sensing, voltage clamp, TRPV4, electrophysiology
1USDA-ARS and Department of Horticulture, University of Wisconsin - Madison, 2Department of Horticulture and Landscape Architecture, Colorado State University
This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.
Published October 14, 2013. Keywords: Chemistry, Technology, Industry, and Agriculture, Life Sciences (General), Potato, amylose, amylopectin, colorimetric assay, iodine
JoVE Immunology and Infection
1Department of Surgery, University of Wisconsin-Madison, School of Medicine and Public Health
We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.
Published May 2, 2013. Keywords: Immunology, Medicine, Molecular Biology, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Cancer Biology, Surgery, Trans-vivo delayed type hypersensitivity, Tv-DTH, Donor antigen, Antigen-specific regulation, peripheral blood mononuclear cells, PBMC, T regulatory cells, severe combined immunodeficient mice, SCID, T cells, lymphocytes, inflammation, injection, mouse, animal model
1School of Environmental and Life Sciences, Kean University, 2Department of Geography, University of Wisconsin-Madison
A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.
Published February 25, 2013. Keywords: Environmental Sciences, Computer Science, Behavior, Infectious Diseases, Geography, Cartography, Data Display, Disease Outbreaks, cartography, human behavior, Trajectory data, space-time activity, GPS, GIS, ArcGIS, spatiotemporal analysis, visualization, segmentation, density surface, density volume, exploratory data analysis, modelling
1Department of Oncology, University of Wisconsin - Madison
A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.
Published December 13, 2012. Keywords: Genetics, Molecular Biology, Cellular Biology, Genomics, DNA synthesis, DNA partitioning, plasmids, lac operator, lac repressor, mammalian cells, fluorescence microscopy
JoVE Immunology and Infection
1Department of Bacteriology, University of Wisconsin-Madison
The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.
Published December 11, 2012. Keywords: Infection, Microbiology, Immunology, Bacteriology, Entomology, Bacteria, injection, pathogenesis, insect larvae, instar, Manduca sexta, tobacco hornworm, animal model, host pathogen interactions
1Department of Bacteriology, University of Wisconsin-Madison
To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.
Published October 19, 2012. Keywords: Microbiology, Molecular Biology, Bacteriology, Developmental Biology, Colonization, Xenorhabdus, Steinernema, symbiosis, nematode, bacteria, fluorescence microscopy
1DOE-Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, 2Department of Soil Science, University of Wisconsin, Madison, 3Department of Soil and Water Science, University of Florida
We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.
Published May 19, 2012. Keywords: Molecular Biology, Glucosamine, muramic acid, microbial residue, aldononitrile acetate derivatization, isotope incorporation, ion structure, electron ionization, GC, MS
JoVE Immunology and Infection
1Wisconsin Water Science Center, United States Geological Survey, 2University of Wisconsin – Madison, 3Agricultural Research Service, United States Department of Agriculture, 4Alaska Science Center, United States Geological Survey
Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.
Published March 3, 2012. Keywords: Immunology, avian influenza virus, environmental sampling, Cryptosporidium, pathogen concentration, Salmonella, water, waterborne disease, waterborne pathogens
1Weldon School of Biomedical Engineering, Purdue University, 2Biomedical Engineering, University of Wisconsin-Madison, 3Biomedical Engineering, University of Michigan, 4Department of Biological Sciences, Purdue University
The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.
Published February 24, 2012. Keywords: Neuroscience, neuroprosthesis, electrode-tissue interface, rejuvenation, neural engineering, neuroscience, neural implant, electrode, brain-computer interface, electrochemistry
1Biomedical Engineering, University of Michigan, 2Biomedical Engineering, University of Wisconsin-Madison, 3NeuroNexus Technologies
We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.
Published February 24, 2012. Keywords: Neuroscience, chronic, silicon electrode, thin film surface electrode, microECoG, surgery, survival, electrophysiology
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Biomedical Engineering, Materials Science Program, Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison
A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues.
Published January 4, 2012. Keywords: Bioengineering, Cell fusion, stem cell, fusogen, cre recombinase, biophotonic imaging, cellular transplantation
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Materials Science Program, University of Wisconsin-Madison, 3Department of Neurological Surgery, University of Wisconsin-Madison, 4Carbone Comprehensive Cancer Center and Center for Stem Cell and Regenerative Medicine, University of Wisconsin-Madison
A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.
Published December 23, 2011. Keywords: Medicine, BTSC, Tumor, cancer stem cell, cell migration, microfluidics, Glioblastoma Multiforme, GBM, chemotaxis, amoeboid, mesenchymal, haptotaxis, PDMS
1Horticulture Department, University of Wisconsin-Madison, 2Department of Zoology, Oregon State University
The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.
Published November 9, 2011. Keywords: Plant Biology, Plant, Arabidopsis thaliana, tomato, 96-well plate, DNA extraction, high-throughput, genotyping
1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California
Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
Published October 27, 2011. Keywords: Molecular Biology, Genetics, mRNA-Seq, Illumina-Seq, gene expression profiling, high throughput sequencing
1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, 2Department of Biochemistry, Waisman Center for Developmental Disabilities, University of Wisconsin
A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.
Published September 17, 2011. Keywords: Neuroscience, synaptoneurosomes, synaptosomes, Percoll-sucrose gradients, neurons, synapse, cortex, mouse
1Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison
Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.
Published August 19, 2011. Keywords: Developmental Biology, Urogenital, prostate, lower urinary tract, urethra, in situ hybridization
1Department of Surgery-Division of Otolaryngology, University of Wisconsin, 2Department of Communicative Disorders, University of Wisconsin
Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.
Published August 8, 2011. Keywords: Neuroscience, ultrasonic vocalization, rat, aging, Parkinson disease, exercise, 6-hydroxydopamine, voice disorders, voice therapy
1Department of Biomedical Engineering, University of Wisconsin – Madison
The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.
Published April 29, 2011. Keywords: Medicine, ex-vivo, mouse, lung, pulmonary vascular impedance, characteristic impedance
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Radiology, University of Wisconsin-Madison
A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.
Published March 7, 2011. Keywords: Medicine, Thermal ablation, interventional oncology, image-guided therapy, radiology, cancer
1Department of Neurological Surgery, School of Medicine and Public Health, University of Wisconsin-Madison
Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.
Published February 5, 2011. Keywords: Neuroscience, Stroke, cerebral ischemia, middle cerebral artery occlusion, intraluminal filament, rat, magnetic resonance imaging, surgery, neuroscience, brain
1Department of Anatomy, University of Wisconsin-Madison
This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.
Published January 24, 2011. Keywords: Neuroscience, hippocampus, cortex, transfection, nucleofection, electroporation, primary culture
1Department of Veterinary Science, University of Wisconsin, Madison, 2Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, 3Present address: Department of Pulmonary Medicine, University of Michigan, Ann Arbor, MI, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI
A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.
Published January 7, 2011. Keywords: Developmental Biology, Zebrafish inner ear, microinjection, electroporation, morpholino
JoVE Immunology and Infection
1Pathology and Laboratory Medicine, University of Wisconsin-Madison, 2Pathobiological Sciences, University of Wisconsin-Madison
Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.
Published November 9, 2010. Keywords: Immunology, CTLs, CD8+ T cells, CFSE labeling, killing assay
JoVE Immunology and Infection
1Department of Medical Microbiology and Immunology, University of Wisconsin
Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.
Published August 12, 2010. Keywords: Microbiology, bone marrow-derived macrophages, fluorescence microscopy, parasitology, Toxoplasma gondii, bradyzoite development, cell culture, cyst wall
1Genetics Training Program, University of Wisconsin-Madison, 2Department of Anatomy, University of Wisconsin-Madison, 3Department of Zoology, University of Wisconsin-Madison, 4Cell and Molecular Biology Training Program, University of Wisconsin-Madison
This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.
Published February 3, 2010. Keywords: Developmental Biology, zebrafish, axon guidance, neural crest, cell behavior, actin, microinjection, embryos
1Department of Chemistry, University of Toronto, 2Department of Chemistry, University of Wisconsin, 3Department of Chemistry, Duke University
The assembly of a nearfield infrared microscope for imaging protein aggregates is described.
Published November 25, 2009. Keywords: Cellular Biology, nearfield imaging, infrared, amyloid, fibril, protein
1Materials Science Program, University of Wisconsin-Madison, 2Department of Biomedical Engineering, University of Wisconsin-Madison
A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.
Published September 2, 2009. Keywords: Biomedical Engineering, automated, passive pumping, microfluidic device, high speed, high flow rate
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Wadsworth Center, New York State Dept. of Health
In this video, we demonstrate the steps required to run a brain-computer interface experiment, including setting up the EEG cap, calibrating the system, and training the user to move a cursor in two dimensions using imagined movements.
Published July 29, 2009. Keywords: Neuroscience, BCI, EEG, brain-computer interface, BCI2000