JoVE General
High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels
1Department of Pharmacology, Vanderbilt University School of Medicine, 2Department of Anesthesiology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine
Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.
JoVE Application Notes
IonFlux: Automated Patch Clamp System with Plate Reader Simplicity - ADVERTISEMENT
The IonFlux Automated Patch Clamp System provides high throughput, cost-effective ion channel screening for a wide range of electrophysiology applications. Fast compound exchange, low cost per data point, and convenient well plate formats make the system ideal for both ligand- and voltage-gated ion channel targets. The IonFlux HT provides an industry-leading 10,000 data points per day, while the IonFlux 16 provides true automated patch clamp performance for about the cost of a manual patch clamp rig.
JoVE General
A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels
A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.
JoVE General
Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies
We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.
JoVE General
Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies
Department of Biology, University of Kentucky, Lexington
We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.
JoVE Applied Physics
Fabrication And Characterization Of Photonic Crystal Slow Light Waveguides And Cavities
School of Physics & Astronomy, University of St Andrews
Use of photonic crystal slow light waveguides and cavities has been widely adopted by the photonics community in many differing applications. Therefore fabrication and characterization of these devices are of great interest. This paper outlines our fabrication technique and two optical characterization methods, namely: interferometric (waveguides) and resonant scattering (cavities).
JoVE General
Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency
1Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University School of Medicine, 2Department of Pharmacology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, 4Research Medicine, Veterans Administration TVHS
We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.
JoVE Neuroscience
Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System
Department of Neurology, Washington University in St. Louis School of Medicine
We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.
JoVE Applied Physics
A Method to Fabricate Disconnected Silver Nanostructures in 3D
1School of Engineering and Applied Sciences, Harvard University, 2Department of Physics, Harvard University
Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm.
JoVE General
High-throughput Screening and Biosensing with Fluorescent C. elegans Strains
1Department of Biology, University of Florida, 2Mount Desert Island Biological Laboratory
A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.
JoVE Neuroscience
Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence
1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California
We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.
JoVE General
Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Department of Physiology and Biophysics, Weill Cornell Medical College
We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.
JoVE Applied Physics
Construction of a High Resolution Microscope with Conventional and Holographic Optical Trapping Capabilities
Department of Physics and Astronomy, University of Utah
The system described herein employs a traditional optical trap as well as an independent holographic optical trapping line, capable of creating and manipulating multiple traps. This allows for the creation of complex geometric arrangements of refractive particles while also permitting simultaneous high-speed, high-resolution measurements of the activity of biological enzymes.
JoVE Neuroscience
Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation
1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, 2Department of Biomedical Engineering, School of Engineering, University of California, Irvine
This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons.
JoVE General
Large Scale Zebrafish-Based In vivo Small Molecule Screen
1Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University School of Medicine, 2Department of Pharmacology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, 4Research Medicine, Veterans Affairs TVHS, Vanderbilt University School of Medicine
Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.
JoVE Neuroscience
Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception
Department of Biology, University of Kentucky
The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.
JoVE General
Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters
Department of Biomedical Engineering, Rutgers University
We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.
JoVE General
A β-glucuronidase (GUS) Based Cell Death Assay
Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.
JoVE Neuroscience
Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking
1Department of Human Development, Cornell University, 2Social Sciences Division, University of Chicago, 3National Brain Research Centre, Manesar, India
Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.
JoVE General
Recapitulation of an Ion Channel IV Curve Using Frequency Components
Bioengineering, University of Utah
There are technical obstacles to measuring current flux through multiple ion channels simultaneously, and later discerning what portion of the transmembrane current is due to each channel type. To address this need, this method presents a way to generate the IV curve of individual channel types using specific frequency components.
JoVE Neuroscience
GABA-activated Single-channel and Tonic Currents in Rat Brain Slices
Department of Neuroscience, Uppsala University, Sweden
We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.
JoVE Clinical and Translational Medicine
In vitro Measurements of Tracheal Constriction Using Mice
Department of Physiology, UT Health Science Center, San Antonio
Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.
JoVE General
Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Faculty of Life Sciences, University of Manchester
Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.
JoVE Neuroscience
Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces
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.
JoVE General
Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis
1Whitehead Institute for Biomedical Research, 2Department of Biology, MIT - Massachusetts Institute of Technology, 3Howard Hughes Medical Institute
SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.
JoVE General
Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes
1Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biomedical Engineering and Cardiac Bioelectricity and Arrhythmia Center, Washington University in St. Louis
Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.
JoVE General
A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning
1MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General Hospital
A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.
JoVE General
High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices
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.
JoVE General
Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes
Department of Physiology and Membrance Biology, University of California, Davis
We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.
JoVE General
Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells
Department of Biology, University of Waterloo
Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).
JoVE Bioengineering
A Microfluidic Device for Studying Multiple Distinct Strains
We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.
JoVE General
Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)
1Center for Biophotonics, University of California, Davis, 2Department of Internal Medicine, University of California, Davis
A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.
JoVE General
In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces
Institute of Biophysics, Johannes Kepler Universitat Linz
This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.
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
Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles
We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.
JoVE General
Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time
1Mechanical Engineering, Johns Hopkins University, 2Biomedical Engineering, Duke University, 3Biomedical Engineering, Johns Hopkins University
We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.
JoVE General
Preparation of Artificial Bilayers for Electrophysiology Experiments
Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University
Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.
JoVE Neuroscience
Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons
1SISSA, International School for Advanced Studies, 2Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 3SISSA Unit, Italian Institute of Technology
Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.
JoVE Bioengineering
Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3
This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.
JoVE General
Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers
Environmental Microfluidics Group, MIT - Massachusetts Institute of Technology
The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.
JoVE General
Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device
1Biomedical Engineering Department, Cornell University, 2Neurosurgical Laboratory for Translational Stem Cell Research, Weill Cornell Brain Tumor Center, Weill Cornell Medical College of Cornell University, 3Cell Morphology Department, Instituto de Investigacion Principe Felipe, 4Department of Chemical and Biomolecular Engineering, Cornell University
We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.
JoVE General
Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes
1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine
This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.
JoVE General
Primary Culture of Adult Rat Heart Myocytes
1Department of Physiology and Cellular Biophysics, Columbia University, 2Department of Pharmacology, Columbia University
In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.
JoVE Neuroscience
Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping
1Wadsworth Center, New York State Department of Health, 2Department of Neurology, Albany Medical College, 3Department of Neurosurgery, Albany Medical College, 4Department of Neurosurgery, Washington University, 5Department of Biomed. Eng., Rensselaer Polytechnic Institute, 6Department of Biomed. Sci., State University of New York at Albany, 7Department of Elec. and Comp. Eng., University of Texas at El Paso
We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.
JoVE General
Genomic Transformation of the Picoeukaryote Ostreococcus tauri
1SynthSys, University of Edinburgh, 2Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris 06, 3UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique, Banyuls-sur-Mer, Université Pierre et Marie Curie, Paris 06
This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.
JoVE General
Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography
Department of Molecular Pharmacology & Therapeutics, Loyola University Chicago
Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.
JoVE Bioengineering
Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows
1Department of Chemical and Biological Engineering, University of Ottawa, 2Department of Mechanical Engineering, University of Ottawa
Micro-particle image velocimetry (μPIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.
JoVE Neuroscience
Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze
Department of Neuroscience, Friedman Brain Institute, Mount Sinai School of Medicine
We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.
JoVE Neuroscience
Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles
Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.
JoVE General
Visualizing Single-molecule DNA Replication with Fluorescence Microscopy
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School
This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.
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