JoVE Neuroscience
Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise
1Department of Biology, University of Kentucky, 2Department of Biological Sciences, Brock University
In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.
JoVE Immunology and Infection
Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions
Department of Medical Microbiology and Immunology, University of Wisconsin
Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.
JoVE Immunology and Infection
Growth of Mycobacterium tuberculosis Biofilms
1Department of Infectious Diseases and Microbiology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh
Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.
JoVE Neuroscience
Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization
Department of Molecular and Cellular Biology, University of Guelph
Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.
JoVE General
Environmentally Induced Heritable Changes in Flax
Department of Biology, Case Western Reserve University
Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.
JoVE Clinical and Translational Medicine
Skeletal Muscle Gender Dimorphism from Proteomics
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College
A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.
JoVE General
LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement
Department of Plant Biology, University of California Davis
Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.
JoVE Editorial
October 2011: This Month in JoVE
Here are some highlights from the October 2011 Issue of Journal of Visualized Experiments (JoVE).
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
Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice
Department of Psychiatry, Brain Research Center, University of British Columbia
The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.
JoVE General
Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays
Department of Biochemistry and Molecular Biology, University of Chicago
We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.
JoVE Neuroscience
Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
Max Planck Institute of Psychiatry
A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.
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 Clinical and Translational Medicine
Protocol for Long Duration Whole Body Hyperthermia in Mice
1Product Development Cell, National Institute of Immunology, 2Small Animal Facility, National Institute of Immunology
This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.
JoVE Neuroscience
A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats
Psychology, University of Toronto Scarborough
Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.
JoVE Clinical and Translational Medicine
An in vivo Assay to Test Blood Vessel Permeability
We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.
JoVE General
Purification of Hsp104, a Protein Disaggregase
Department of Biochemistry and Biophysics, University of Pennsylvania
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
JoVE General
High-throughput Yeast Plasmid Overexpression Screen
1Neuroscience Graduate Group, University of Pennsylvania School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine
Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.
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 General
Monitoring Plant Hormones During Stress Responses
Department of Biology, University of Texas
A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.
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
Assay for Adhesion and Agar Invasion in S. cerevisiae
Department of Molecular Biology, Princeton University
We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.
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 Clinical and Translational Medicine
Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test
Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic
Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice.
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
Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons
Department of Molecular Pharmacology and Experimental Therapeutics, Loyola University Chicago
We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.
JoVE General
Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates
1Department of Chemical Physiology, The Scripps Research Institute, 2Department of Molecular and Experimental Medicine, The Scripps Research Institute
In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.
JoVE General
Microarray Analysis for Saccharomyces cerevisiae
Vermont Genetics Network, The University of Vermont
In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.
JoVE General
Solid Plate-based Dietary Restriction in Caenorhabditis elegans
1Department of Internal Medicine, Division of Geriatric Medicine, University of Michigan, 2Department of Molecular and Integrative Physiology, University of Michigan
Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.
JoVE General
4D Imaging of Protein Aggregation in Live Cells
Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.
JoVE Clinical and Translational Medicine
A Zebrafish Model of Diabetes Mellitus and Metabolic Memory
1Dr. William M. Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science, 2Chicago Medical School, Rosalind Franklin University of Medicine and Science
Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.
JoVE Clinical and Translational Medicine
Implantation of Radiotelemetry Transmitters Yielding Data on ECG, Heart Rate, Core Body Temperature and Activity in Free-moving Laboratory Mice
1Division of Surgical Research, University Hospital Zurich, 2Institute of Laboratory Animal Science, University of Zurich
A surgical technique for implantation of commercially available telemetry transmitters used for continuous measurement of biopotential (one-lead ECG), heart rate, core body temperature and locomotor activity in freely moving mice is shown. Recommendations and protocols for post-operative care and pain relief, improving recovery, well being and survival rate are also presented.
JoVE Neuroscience
The Tail Suspension Test
1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3The Program in Neuroscience, University of Maryland, 4Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine
The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.
JoVE Immunology and Infection
Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions
Department of Physiology and Pharmacology, University of Calgary
This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.
JoVE General
Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
Department of Biology, University of North Carolina at Charlotte
Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.
JoVE Neuroscience
Operant Sensation Seeking in the Mouse
In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.
JoVE General
Fluorescence Activated Cell Sorting of Plant Protoplasts
Center for Genomics and Systems Biology, Department of Biology, New York University
A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.
JoVE Neuroscience
Methods to Assay Drosophila Behavior
1Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, 2Department of Genetics, Louisiana State University Health Sciences Center
Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.
JoVE General
Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture
1Biology Department, Western Washington University, 2Washington State University Northwestern Research and Extension Center, 3Department of Plant and Soil Science, Texas Tech University
Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.
JoVE Immunology and Infection
Quantitative Measurement of the Immune Response and Sleep in Drosophila
Center for Sleep and Circadian Neurobiology, University of Pennsylvania Perelman School of Medicine
To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.
JoVE Bioengineering
Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy
1Centre for Vascular Research and Australian Centre for Nanomedicine, The University of New South Wales, 2School of Chemistry and Australian Centre for Nanomedicine, The University of New South Wales
A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.
JoVE General
In Situ Hybridization for the Precise Localization of Transcripts in Plants
The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.
JoVE General
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich
Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.
JoVE Immunology and Infection
In vitro Biofilm Formation in an 8-well Chamber Slide
Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital
This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide
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
A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development
Department of Microbiology and Immunology, Loyola University Medical Center
We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.
JoVE General
Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment
1School of Earth Sciences, The Ohio State University, 2School of Environment & Natural Resources, The Ohio State University, 3Institute of Geology and Geophysics, Chinese Academy of Sciences
We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.
JoVE General
Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics
Department of Marine Sciences, University of Georgia (UGA)
We present a method for generating cDNA from environmental mRNA. In general, total RNA is first collected from the environment, rRNA is selectively removed, mRNA is selectively amplified, and cDNA synthesized from the enriched mRNA pool is sequenced. Recovered sequences can be annotated using standard bioinformatics techniques to identify the expressed genes.
JoVE Clinical and Translational Medicine
Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR
1Department of Surgery, UCSF/VA Medical Center, 2Clinical & Regulatory, LoneStar Heart, Inc.
This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.
JoVE Clinical and Translational Medicine
The Trier Social Stress Test Protocol for Inducing Psychological Stress
Department of Psychology, Northern Arizona University
This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.
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