28 articles published in JoVE
1Department of Physiology and Biophysics, University of Washington
Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.
Published June 21, 2013. Keywords: Physiology, Biophysics, Molecular Biology, Biochemistry, Genetics, Cellular Biology, Proteins, Membranes, Artificial, Lipid Bilayers, Liposomes, Phospholipids, biochemistry, Lipids, Giant Unilamellar Vesicles, liposome, electrophysiology, electroformation, reconstitution, patch clamp
1Department of Biology, University of Washington
Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.
Published February 24, 2013. Keywords: Neuroscience, Neurobiology, Physiology, Biochemistry, Chemistry, Entomlogy, Behavior, electrophysiology, olfaction, olfactory system, insect, multi-channel recording, gas chromatography, pollination, bees, Bombus impatiens, antennae, brain, animal model
1Department of Genome Sciences, University of Washington
We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.
Published February 23, 2013. Keywords: Genetics, Molecular Biology, Microbiology, Biochemistry, Cellular Biology, Basic Protocols, Genomics, Eukaryota, Bacteria, Biological Phenomena, Metabolic Phenomena, Genetic Phenomena, Microbiological Phenomena, Life sciences, chemostat, evolution, experimental evolution, Ministat, yeast, E. coli., Physiology, Continuous culture, high throughput, arrays, cell culture
1Department of Biology, University of Washington, 2Howard Hughes Medical Institute, University of Washington, 3PRESTO, Japan Science and Technology Agency
We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.
Published December 31, 2012. Keywords: Plant Biology, Molecular Biology, Developmental Biology, Cellular Biology, Botany, plant, live imaging, epidermis, stomata, confocal, time lapse, Arabidopsis, cotyledon
1School of Environmental and Forest Sciences, University of Washington, 2Center for Agricultural and Rural Development, Department of Economics, Iowa State University, 3Department of Civil, Architectural, and Environmental Engineering, North Carolina A&T University, 4Iowa Geological and Water Survey
This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs.
Published December 9, 2012. Keywords: Environmental Sciences, Plant Biology, Civil Engineering, Forest Sciences, Water quality, multiobjective optimization, evolutionary algorithms, cost efficiency, agriculture, development
1Department of Speech & Hearing Sciences, Institute for Learning and Brain Sciences, University of Washington
We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.
Published October 24, 2012. Keywords: Neuroscience, Magnetoencephalography, MEG, Electroencephalography, EEG, audition, attention, inverse imaging
1Department of Anesthesiology & Pain Medicine, Mitochondria and Metabolism Center, University of Washington, Seattle
A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.
Published September 7, 2012. Keywords: Cellular Biology, Mitochondria, respiration, mitochondrial permeability transition pore (mPTP), membrane potential, swelling, calcium, spectrofluorometer
1Biomedical Engineering, University of Michigan, 2Department of Neurological Surgery, University of Washington School of Medicine
Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.
Published July 30, 2012. Keywords: Neuroscience, Physiology, Biomedical Engineering, Paraformaldehyde, fixation, physiological pressures, vascular system, brain, preservation
1Department of Biochemistry, University of Washington, 2Molecular and Cellular Biology Program, University of Washington
This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems
Published July 20, 2012. Keywords: Biochemistry, Molecular Biology, Cellular Biology, Genetics, Developmental Biology, C. elegans, hypoxia, hypoxia inducible factor-1 (hif-1), anoxia, oxygen
1Department of Pathology, Center for Cardiovascular Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, 2Departments of Bioengineering and Medicine/Cardiology, University of Washington
Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (μCT) imaging.
Published February 10, 2012. Keywords: Medicine, Vascular biology, heart, coronary vessels, mouse, micro Computed Tomography (μCT) imaging, Microfil
1Buck Institute for Research on Aging, 2Department of Physiology & Biophysics, University of Washington
Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.
Published December 28, 2011. Keywords: Molecular Biology, Single cell analysis, Microarray, Gene expression, Cardiomyocyte, Mouse heart perfusion, mice, qPCR
1Department of Biochemistry, Temple University, 2Department of Anesthesiology and Pain Medicine, University of Washington
An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.
Published December 21, 2011. Keywords: Molecular Biology, Reactive oxygen species, Calcium, paracrine superoxide, endothelial cells, confocal microscopy
1Electrical Engineering Department, University of Washington, 2Division of Human Biology, Fred Hutchinson Cancer Research Center, 3Molecular and Cellular Biology Program, University of Washington, 4Clinical Research, Fred Hutchinson Cancer Research Center, 5Public Health Sciences, Fred Hutchinson Cancer Research Center
Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.
Published September 27, 2011. Keywords: Bioengineering, Surface plasmon, optical trapping, optical tweezers, plasmonic trapping, cell manipulation, optical manipulation
1College of Nursing, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 2College of Science and Engineering, Interdisciplinary Life Sciences Research Laboratory, Seattle University, 3School of Medicine, University of Washington
An in vitro method for preparing functional glucocorticoid receptor (GR)•hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.
Published September 21, 2011. Keywords: Biochemistry, glucocorticoid receptor, hsp90, molecular chaperone protein, in vitro reconstitution, steroid binding, biochemistry, immunoadsorption, immunoprecipitation, Experion, western blot
JoVE Immunology and Infection
1Research Animal Diagnostic Services, Charles River, 2Research Models and Services, Charles River, 3Department of Comparative Medicine, University of Washington
This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.
Published September 6, 2011. Keywords: Immunology, rat, mouse, endoparasite, ectoparasite, diagnostics, mites, pinworm, helminths, protozoa, health monitoring
1Research Animal Diagnostic Services, Charles River, 2Research Models and Services, Charles River, 3Department of Comparative Medicine, University of Washington
This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.
Published August 7, 2011. Keywords: Anatomy, rodent, necropsy, diagnostic assay, bacteriology, PCR, organ collection, tissue sampling
1Department of Otolaryngology-Head and Neck Surgery, Virginia Merrill Bloedel Hearing Research Center, University of Washington, 2Department of Physiology and Biophysics, Virginia Merrill Bloedel Hearing Research Center, University of Washington
The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.
Published March 21, 2011. Keywords: Neuroscience, slice preparation, chicken auditory brainstem, organotypic cultures, nucleus laminaris, nucleus magnocellularis
1Department of Physiology and Biophysics, University of Washington School of Medicine
We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.
Published February 3, 2011. Keywords: Neuroscience, Hippocampus, Hippocampal formation, Brain Slices, Organotypic Cultures, Synaptic Transmission, Synaptic Physiology
1Department of Anesthesiology & Pain Medicine, University of Washington School of Medicine
Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.
Published August 31, 2010. Keywords: Medicine, cardiac physiology, high energy phosphate, phosphocreatine, ATP
1Department of Biochemistry, University of Washington, 2Department of Neurology, University of Washington, 3Division of Genetics, Departments of Pediatrics and Cellular and Molecular Medicine, and the Institute for Genomic Medicine, University of California, San Diego - Rady Children’s Hospital
We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.
Published May 21, 2010. Keywords: JoVE Neuroscience, Neurodegeneration, Mouse behavior assay, cerebellar ataxia, polyglutamine disease
1Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine
Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain.
Published January 27, 2010. Keywords: JoVE Medicine, diffuse noxious inhibitory control, DNIC, temporal summation, TS, psychophysical testing, endogenous analgesia, pain modulation, pregnancy-induced analgesia, cesarean section, post-operative pain, prediction
1Department of Radiology, University of Washington, 2Department of Bioengineering, University of Washington
This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.
Published October 20, 2009. Keywords: Medicine, Small animal, MRI, MRS, mouse, brain, skeletal muscle, tumor, ischemia
1Department of Biochemistry, University of Washington, 2Department of Pathology, University of Washington
In this article we present a general protocol for measuring the replicative life span of yeast mother cells.
Published June 25, 2009. Keywords: Developmental Biology, aging, longevity, life span, yeast, dietary restriction, Saccharomyces cerevisiae
1Department of Pathology, University of Washington, 2Molecular and Cellular Biology Program, University of Washington
In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.
Published May 12, 2009. Keywords: Developmental Biology, Caenorhabditis elegans, aging, longevity, life span assay, worms, nematode, dietary restriction, RNA interference
1Department of Pathology, University of Washington
Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.
Published May 6, 2009. Keywords: Microbiology, longevity, aging, chronological life span, yeast, Bioscreen C MBR, stationary phase
1Department of Bioengineering, University of Pennsylvania, 2University of Washington
In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.
Published October 1, 2007. Keywords: Cellular biology, mechanotransduction, traction force, microfabrication
1Dept. of Bioengineering, University of Washington
We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.
Published October 1, 2007. Keywords: Cellular Biology, BioMEMs, Microvalves, Soft lithography, PDMS, Parallel Mixer, Integrated microfluidic system, Herringbone mixer, Diffusion Gradients, Bioengineering
1Department of Biomedical Engineering, University of Washington
The ability to culture cells has revolutionized hypothesis testing in basic cell and molecular biology research. It has become a standard methodology in drug screening, toxicology, and clinical assays, and is increasingly used in regenerative medicine. However, the traditional cell culture methodology essentially consisting of the immersion of a large population of cells in a homogeneous fluid medium and on a homogeneous flat substrate has become increasingly limiting both from a fundamental and practical perspective. Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, and the medium composition, as well as the neighboring cell type in the surrounding cellular microenvironment. Additionally, microtechnology is conceptually well-suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. In this interview, Albert Folch explains these limitations, how they can be overcome with soft lithography and microfluidics, and describes some relevant examples of research in his lab and future directions.
Published October 1, 2007. Keywords: Biomedical Engineering, BioMEMS, Soft Lithography, Microfluidics, Agrin, Axon Guidance, Olfaction, Interview