JoVE Editorial
May 2011: This Month in JoVE
The main highlights for our May issue include methods for measuring cognition in zero gravity, isolating mosquito immune cells, engineering recombinant SARS vaccines, and detecting tumors with thermal imaging. In addition, procedures for isolating neural stem cells from human fetal brain and culturing antigen-presenting liver cells will also be released.
JoVE Neuroscience
Coherence between Brain Cortical Function and Neurocognitive Performance during Changed Gravity Conditions
1Institute of Movement and Neurosciences, German Sport University Cologne, 2Deptartment of Surgical Skills, University of Toronto, 3School of Human Movement Studies, Institute of Health and Biomedical Innovation, Queensland University of Technology, 4Brain Products GmbH, Scientific Support, Gilching, Germany
The effect of weightlessness and hypergravity on both hemodynamic and electrophysiological processes in the brain is going to be followed during parabolic flight by EEG and NIRS techniques. A feasibility study of a more complex experiment, which is planned to carry out during medium- and long-term space flight.
JoVE General
Operant Learning of Drosophila at the Torque Meter
Department of Neurobiology, Free University of Berlin
Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.
JoVE Neuroscience
Vertical T-maze Choice Assay for Arthropod Response to Odorants
Entomology and Nematology Department, University of Florida
A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.
JoVE General
Visually Mediated Odor Tracking During Flight in Drosophila
Department of Physiological Science, University of California, Los Angeles
Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.
JoVE Neuroscience
Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection
Department of Biological Sciences, Florida Atlantic University
An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.
JoVE Neuroscience
Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster
Department of Neurobiology, Stanford University
Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.
JoVE Neuroscience
Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster
1Institute of Healthy Ageing, and GEE, University College London - UCL, 2School of Biosciences, University of Kent
The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.
JoVE Neuroscience
Preparation of Drosophila Central Neurons for in situ Patch Clamping
School of Life Sciences, Arizona State University
In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.
JoVE Bioengineering
Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer
The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed
JoVE General
Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles
Gene Expression and Signaling Research Group, Max Planck Institute for Biophysical Chemistry
Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.
JoVE General
A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila
Department of Physiological Science, University of California, Los Angeles
Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.
JoVE Applied Physics
Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation
Chemical Sciences Division, Lawrence Berkeley National Laboratory
A molecular beam coupled to tunable vacuum ultraviolet photoionization mass spectrometer at a synchrotron provides a convenient tool to explore the electronic structure of isolated gas phase molecules and clusters. Proton transfer mechanisms in DNA base dimers were elucidated with this technique.
JoVE Clinical and Translational Medicine
Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)
1Division of Cardiovascular Medicine, University of Pennsylvania, Perelman School of Medicine, 2Department of Radiology, University of Pennsylvania, Perelman School of Medicine, 3Department of Dermatology, University of Pennsylvania, Perelman School of Medicine
There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.
JoVE Neuroscience
Studying the Neural Basis of Adaptive Locomotor Behavior in Insects
Zoological Institute, University of Cologne
We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.
JoVE Immunology and Infection
Isolation of Mouse Lung Dendritic Cells
Pathobiological Sciences, Louisiana State University
A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.
JoVE Neuroscience
Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System
1Department of Biological Sciences, Florida Atlantic University, 2Department of Chemistry & Biochemistry, Florida Atlantic University
A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.
JoVE General
Measures of Heart and Ventilatory Rates in Freely Moving Crayfish
Department of Biology, University of Kentucky
Invertebrates show an autonomic sympathetic-like response similar to that described for vertebrates. The coordination of the cardio-vascular and ventilatory systems allows for measurement of a biological index in which to assess an organism internal state.
JoVE Neuroscience
Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)
Fachbereich Bio/Chem/Pharm, Institut für Biologie – Neurobiologie, Freie Universität Berlin
We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.
JoVE General
An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice
1Physical Therapy Department, University of Missouri, Columbia, 2Biomedical Sciences Department, University of Missouri, Columbia
We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.
JoVE General
An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Wellcome Trust Centre for Human Genetics, University of Oxford
Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.
JoVE Bioengineering
Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering
1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 2Biomedical Sciences Program, University of California, San Diego, 3Department of Bioengineering, University of California, San Diego
Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues
JoVE Applied Physics
Bringing the Visible Universe into Focus with Robo-AO
1Caltech Optical Observatories, California Institute of Technology, 2Department of Astronomy, California Institute of Technology, 3Dunlap Institute for Astronomy and Astrophysics, University of Toronto, 4Inter-University Centre for Astronomy & Astrophysics, 5Observatories of the Carnegie Institution for Science, 6Benoziyo Center for Astrophysics, Weizmann Institute of Science
Light from astronomical objects must travel through the earth's turbulent atmosphere before it can be imaged by ground-based telescopes. To enable direct imaging at maximum theoretical angular resolution, advanced techniques such as those employed by the Robo-AO adaptive-optics system must be used.
JoVE General
Analyzing Large Protein Complexes by Structural Mass Spectrometry
Department of Biological Chemistry, Weizmann Institute of Science
Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.
JoVE Clinical and Translational Medicine
Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers
1Thayer School of Engineering, Dartmouth College, 2Department of Physics and Astronomy, Dartmouth College, 3Darmouth Medical School, Dartmouth College, 4School of Computer Science, University of Birmingham
Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.
JoVE Neuroscience
An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces
Department of Bioengineering, Imperial College London
We use a closed-loop fly-machine interface to investigate general principles in neuronal control.
JoVE Applied Physics
Atom Probe Tomography Studies on the Cu(In,Ga)Se2 Grain Boundaries
1Department of Microstructure Physics and Alloy Design, Max-Planck-Institut für Eisenforschung GmbH, 2Zentrum für Sonnenenergie- und Wasserstoff-Forschung Baden-Württemberg ( ZSW )
In this work, we describe the use of the atom-probe tomography technique for studying the grain boundaries of the absorber layer in a CIGS solar cell. A novel approach to prepare the atom probe tips containing the desired grain boundary with a known structure is also presented here.
JoVE General
Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes
1Experimental and Clinical Research Center (ECRC), Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC), 2Medical Department, Division of Cardiology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, 3Medical Department, Division of Cardiology and Angiology, Campus Mitte, Charité - Universitätsmedizin Berlin
Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.
JoVE General
OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides
1Energy Biosciences Institute, University of California, Berkeley, 2Department of Plant and Microbial Biology, University of California, Berkeley
A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.
JoVE General
Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans
Centre d’Immunologie de Marseille-Luminy, Université de la Méditerranée
We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.
JoVE Bioengineering
Polymer Microarrays for High Throughput Discovery of Biomaterials
1Laboratory of Biophysics and Surface Analysis, University of Nottingham, 2School of Molecular Medical Sciences, University of Nottingham, 3David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.
JoVE General
GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil
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.
JoVE Neuroscience
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
1Department of Neurology and Committee on Neurobiology, The University of Chicago Medical Center, 2Department of Neurology, The University of Chicago Medical Center
Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.
JoVE Neuroscience
MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions
1Department of Human Perception, Cognition and Action, Max Planck Institute for Biological Cybernetics, 2Laboratoire de Physiologie de la Perception et de l'Action, Collège de France - CNRS, 3Department of Brain and Cognitive Engineering, Korea University
An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.
JoVE Chemistry
Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
1Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 2The University of Melbourne
Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.
JoVE General
Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure
Department of Chemistry, George Washington University
Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.
JoVE Clinical and Translational Medicine
Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice
1European Institute for Molecular Imaging, Westfälische Wilhelms-University Münster, 2British Heart Foundation Cardiovascular Sciences Unit, Imperial College London, 3Department of Bioengineering, Imperial College London, 4Biomedical Engineering, Eindhoven University of Technology
The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.
JoVE General
Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential
Department of Microbiology, Miami University
Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.
JoVE General
Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water
Boston Biomedical Research Institute
Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.
JoVE General
T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Department of Biological Chemistry, Weizmann Institute of Science
Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.
JoVE Immunology and Infection
Mass Spectrometric Analysis of Glycosphingolipid Antigens
1Undergraduate Program, Rice University, 2Proteomics Facility, Department of Pathology, University of Texas MD Anderson Cancer Center, 3Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, 4University of Texas Graduate School of Biological Sciences at Houston
A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.
JoVE Clinical and Translational Medicine
MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease
1Department of Pharmaceutical Biosciences, Uppsala University, 2Department of Chemical and Biological Engineering, Chalmers University of Technology
Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson’s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.
JoVE Bioengineering
Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins
Department of Biological Sciences, University of North Texas
This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.
JoVE Chemistry
Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)
Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.
JoVE General
Bioassays for Monitoring Insecticide Resistance
1Division of Plant Sciences, University of Missouri, Delta Research Center, 2Department of Entomology, Louisiana State University Agricultural Center
This manuscript demonstrates and discusses techniques used to survey pesticide susceptibility and detect resistance to contact and systemic pesticides in arthropod pests.
JoVE General
Measuring Caenorhabditis elegans Life Span on Solid Media
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.
JoVE Applied Physics
Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments
1Materials Science Division, Argonne National Laboratory, 2Energy Systems Division, Argonne National Laboratory, 3MassThink LLC
White light microscope interferometry is an optical, noncontact and quick method for measuring the topography of surfaces. It is shown how the method can be applied toward mechanical wear analysis, where wear scars on tribological test samples are analyzed; and in materials science to determine ion beam sputtering or laser ablation volumes and depths.
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