Research Design:
A plan for collecting and utilizing data so that desired information can be obtained with sufficient precision or so that an hypothesis can be tested properly.
JoVE General
Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
1Plant Molecular and Cellular Biology Program, University of Florida, 2Department of Biology, University of Florida, 3Interdisciplinary Center for Biotechnology Research, University of Florida, 4Genetics Institute, University of Florida
Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.
JoVE Chemistry
Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)
Proteomics and Metabolomics Facility, Colorado State University
Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.
JoVE Neuroscience
Brain Imaging Investigation of the Neural Correlates of Emotional Autobiographical Recollection
1Department of Psychiatry, University of Alberta, Edmonton, 2Psychology Department, University of Illinois, Urbana-Champaign, 3Neuroscience Program, University of Illinois, Urbana-Champaign, 4Beckman Institute for Advanced Science & Technology, University of Illinois, Urbana-Champaign
We present a protocol that allows investigation of the neural correlates of recollecting emotional autobiographical memories, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.
JoVE Neuroscience
The Mouse Forced Swim Test
1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 4The Program in Neuroscience, University of Maryland
The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.
JoVE General
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.
JoVE Neuroscience
Brain Imaging Investigation of the Neural Correlates of Observing Virtual Social Interactions
1Department of Computing Science, University of Alberta, 2Department of Psychology, University of Illinois, 3Centre for Neuroscience, University of Alberta, 4Department of Psychology, University of Alberta, 5Department of Marketing, Business Economics, and Law, University of Alberta, 6Neuroscience Program, University of Illinois at Urbana-Champaign, 7Beckman Institute, University of Illinois at Urbana-Champaign
This article demonstrates an experimental design in which whole-body animated characters are used in conjunction with functional magnetic resonance imaging (fMRI) to investigate the neural correlates of observing virtual social interactions.
JoVE Neuroscience
Brain Imaging Investigation of the Neural Correlates of Emotion Regulation
1Department of Psychology, University of Illinois, Urbana-Champaign, 2Department of Computing Science, University of Alberta, Edmonton, 3Department of Psychiatry, University of Alberta, Edmonton, 4Department of Psychology, University of Alberta, Edmonton, 5Centre for Neuroscience, University of Alberta, Edmonton, 6Neuroscience Program, University of Illinois, Urbana-Champaign, 7Beckman Institute, University of Illinois, Urbana-Champaign
We present a protocol that allows investigation of the neural correlates of deliberate and automatic emotion regulation, using functional magnetic resonance imaging. This protocol can be used in healthy participants, both young and older, as well as in clinical patients.
JoVE Bioengineering
GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization
1DOE Plant Research Laboratory, Michigan State University, 2DOE Great Lakes Bioenergy Research Center, Michigan State University
GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.
JoVE General
Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors
Institute for Biological Interfaces, Forschungszentrum Karlsruhe
We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.
JoVE Neuroscience
Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord
1Département Nociception et Douleur, Institut des Neurosciences Cellulaires et Intégratives, Centre National de la Recherche Scientifique (CNRS), 2Departments of Anesthesiology and Pharmacology, Columbia University, 3Department of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences
Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.
JoVE General
Anterior Cervical Discectomy and Fusion in the Ovine Model
1Monash Immunology and Stem Cell Laboratories (MISCL), Monash University, 2Department of Surgery, Monash University
This video demonstrates the technique of anterior cervical discectomy and fusion in the ovine model.
JoVE General
Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique
1Department of Molecular Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, 2Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw
DT40, a model vertebrate genetic system, provides a powerful tool to analyze protein function. Here we describe a simple method that allows qualitative analysis of parameters that influence DNA synthesis during the S-phase in DT40 cells at the single molecule level.
JoVE General
Measurement of Lifespan in Drosophila melanogaster
1Department of Molecular and Integrative Physiology, University of Michigan, 2Cellular and Molecular Biology Program, University of Michigan
Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.
JoVE General
How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)
1Department of Neurology, Emory University School of Medicine, 2Coulter Department of Biomedical Engineering, Laboratory for Neuroengineering, Georgia Institute of Technology and Emory, University School of Medicine, 3Emory University School of Medicine
This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.
JoVE General
Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency
Gene Expression Division, Bio-Rad Laboratories, Inc.
This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.
JoVE Immunology and Infection
Murine Superficial Lymph Node Surgery
1Maisonneuve-Rosemont Hospital Research Center, 2Department of Microbiology and Immunology, University of Montreal, 3Department of Medicine, University of Montreal
To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.
JoVE General
Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University
Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).
JoVE Clinical and Translational Medicine
Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring
Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.
JoVE General
Generation of Mice Derived from Induced Pluripotent Stem Cells
1Dorris Neuroscience Center & Department of Cell Biology, The Scripps Research Institute, 2Mouse Genetics Core Facility, The Scripps Research Institute
Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.
JoVE Neuroscience
Manual Drainage of the Zebrafish Embryonic Brain Ventricles
We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.
JoVE Clinical and Translational Medicine
Use of a Hanging-weight System for Liver Ischemia in Mice
1UCH Transplant Center, University of Colorado, Denver, 2Department of Anesthesiology, University of Colorado, Denver
We established a novel murine model of a hanging weight system for portal triad occlusion. This technique may be useful for future investigations of ischemia in murine hepatic models.
JoVE Neuroscience
Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition
1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University, 5Department of Psychiatry and Behavioral Sciences, Duke University, 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois
We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.
JoVE Neuroscience
Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography (dEEG)
Department of Psychology, University Toronto Scarborough
Dense array electroencephalography is being used increasingly to study social cognitive functions in infants and adults. Here we present an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults.
JoVE General
Fabrication and Use of MicroEnvironment microArrays (MEArrays)
1Life Science Division, Lawrence Berkeley National Laboratory, 2Department of Comparative Biochemistry, University of California, Berkeley
A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.
JoVE Immunology and Infection
The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation
Department of Immunology, John Curtin School of Medical Research, Australian National University
CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.
JoVE Clinical and Translational Medicine
Cecal Ligation Puncture Procedure
1Department of Microbiology and Immunology School of Medicine, Temple University, 2Department of Biochemistry, School of Medicine, Temple University
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
JoVE Bioengineering
Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart
Department of Biomedical Engineering, Washington University in St. Louis
This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.
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 Neuroscience
Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution
Section of Neurobiology, Center for Learning and Memory, The University of Texas at Austin
Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.
JoVE General
Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
1School of Forestry and Environmental Studies, Yale University, 2Department of Biological Sciences, Virginia Tech, 3Department of Ecology, Evolution and Behavior, The Hebrew University of Jerusalem
We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.
JoVE Neuroscience
Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)
In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.
JoVE Neuroscience
Brain Imaging Investigation of the Memory-Enhancing Effect of Emotion
1Centre for Neuroscience, University of Alberta, 2Neuroscience Program, University of Illinois, Urbana-Champaign, 3Center for Cognitive Neuroscience, Duke University, 4Psychology Department, Neuroscience Program, & Beckman Institute, University of Illinois, Urbana-Champaign
We present a protocol that uses functional magnetic resonance imaging to investigate the neural correlates of the memory-enhancing effect of emotion. This protocol allows identification of brain activity specifically linked to memory-related processing, contrary to more general perceptual processing, and can be used with healthy and clinical populations.
JoVE Immunology and Infection
Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System
Department of Immunology, College of Medicine, Mayo Clinic
The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.
JoVE Neuroscience
Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example
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.
JoVE Immunology and Infection
Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)
Blood Research Institute, BloodCenter of Wisconsin
For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.
JoVE Bioengineering
Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
Biomedical Engineering, Tulane University
Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.
JoVE Neuroscience
The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice
1Institute for Neuroscience, University of Texas at Austin, 2Department of Psychology, University of Texas at Austin, 3Department of Neurology, University of Florida, 4Department of Psychiatry, University of Texas Southwestern Medical Center, 5Department of Neuroscience, McKnight Brain Institute, University of Florida
The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.
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 Neuroscience
Peering into the Dynamics of Social Interactions: Measuring Play Fighting in Rats
Department of Neuroscience, University of Lethbridge
Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.
JoVE Clinical and Translational Medicine
Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children
1Department of Public Health, Academic Medical Center - University of Amsterdam, 2Department of Epidemiology, Documentation and Health Promotion, Public Health Service of Amsterdam (GGD), 3Department of Biological Psychology, VU University, 4EMGO+ Institute, VU University Medical Center, 5Institute of Health Sciences, VU University, 6Department of Pediatrics, VU University Medical Center
Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.
JoVE Bioengineering
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
1The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, 2Center for Reproductive Sciences, University of California San Francisco, 3Department of Urology, University of California San Francisco, 4Department of Cell and Tissue Biology, University of California San Francisco, 5Fluidigm Corporation, Fluidigm Corporation, 6Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, 7UCSF - Helen Diller Family Comprehensive Cancer Center, University of California San Francisco
Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.
JoVE Clinical and Translational Medicine
Use of a Hanging-weight System for Isolated Renal Artery Occlusion
1Department of Anesthesiology, University of Colorado, 2School of Medicine, University of Colorado, 3Department of Anesthesiology, Korea University College of Medicine
A precise murine model for acute kidney injury (AKI) due to ischemia is an important tool to investigate acute kidney injury and possibly find therapeutic tools to treat renal injury. The hanging weight system offers a tool for immediate and reliable renal artery occlusion and reperfusion without causing renal congestion.
JoVE General
A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities
Department of Molecular and Cell Biology, Harvard
We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.
JoVE General
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.
JoVE Clinical and Translational Medicine
Orthotopic Liver Transplantation in Rats
1Transplantation Division, Department of Surgery, University of Geneva Hospitals, 2Department of Surgery, University of Pavia, 3Department of Surgery, University of Geneva, 4Division of Abdominal Surgery, Department of Surgery, University of Geneva Hospitals
We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.
JoVE General
Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells
Department 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.
JoVE General
Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth
1UCR Stem Cell Center, University of California, 2Department of Cell Biology and Neuroscience, University of California, 3Cell, Molecular, and Developmental Biology Graduate Program, University of California, 4Center for Research in Intelligent Systems, University of California
Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.
JoVE Neuroscience
Novel Apparatus and Method for Drug Reinforcement
College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin
Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.
JoVE General
An Introduction to Worm Lab: from Culturing Worms to Mutagenesis
Department of Biology, University of Texas at Arlington
Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.
JoVE Immunology and Infection
Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection
Department of Microbiology, Immunology and Pathology, Colorado State University
Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.
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