JoVE: Journal of Visualized Experiments

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Mon, 08 Feb 2010 17:30:00 EST

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Depletion of Specific Cell Populations by Complement Depletion

Fri, 05 Feb 2010 14:00:00 EST

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

Thu, 04 Feb 2010 00:00:00 EST

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Wed, 03 Feb 2010 16:25:00 EST

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Using the optokinetic response to study visual function of zebrafish

Tue, 02 Feb 2010 18:45:00 EST

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults^1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Mon, 01 Feb 2010 16:25:00 EST

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Fri, 29 Jan 2010 00:00:00 EST

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Isolation of Mouse Peritoneal Cavity Cells

Thu, 28 Jan 2010 16:45:00 EST

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)

Wed, 27 Jan 2010 16:25:00 EST

Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain.

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

Tue, 26 Jan 2010 17:00:00 EST

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Mon, 25 Jan 2010 16:30:00 EST

This video describes a right hemihepatectomy with intrahepatic transection of the right hemipedicle leaving the hepatoduodenal ligament completely untouched.

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Fri, 22 Jan 2010 16:52:00 EST

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Thu, 21 Jan 2010 00:00:00 EST

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Erratum: Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

Wed, 20 Jan 2010 23:57:00 EST

A correction to author names has been made for article Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease

Wed, 20 Jan 2010 18:15:00 EST

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Tue, 19 Jan 2010 17:00:00 EST

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Spinal Cord Electrophysiology

Mon, 18 Jan 2010 16:00:00 EST

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Fri, 15 Jan 2010 00:00:00 EST

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Visualizing RNA Localization in Xenopus Oocytes

Thu, 14 Jan 2010 17:00:00 EST

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

CoolCell - Controlled Cell Freezing w/o Alcohol or Maintenance Cost

Thu, 14 Jan 2010 00:00:00 EST

The 2009 Lindau Nobel Laureate Meeting: Roger Y. Tsien, Chemistry 2008

Wed, 13 Jan 2010 16:01:00 EST

American biochemist Roger Tsien shared the 2008 Nobel Prize in Chemistry with Martin Chalfie and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Tsien dramatically improved the wild-type GFP resulting in increased fluorescence, increased photostability, and a shift in the major excitation peak to 488 nm (matching FITC).

A Protocol for the Production of KLRG1 Tetramer

Tue, 12 Jan 2010 16:01:00 EST

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

In vivo-like Organotypic Murine Retinal Wholemount Culture

Mon, 11 Jan 2010 17:30:00 EST

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Fri, 08 Jan 2010 17:00:00 EST

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Thu, 07 Jan 2010 00:00:00 EST

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.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Wed, 06 Jan 2010 17:10:00 EST

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Tue, 05 Jan 2010 20:00:00 EST

We will show how to record flash responses from single mouse cones using a suction electrode.

Presynaptically Silent Synapses Studied with Light Microscopy

Mon, 04 Jan 2010 17:00:00 EST

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

Thu, 31 Dec 2009 14:00:00 EST

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Wed, 30 Dec 2009 15:00:00 EST

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Tue, 29 Dec 2009 15:00:00 EST

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Engineering Cell-permeable Protein

Mon, 28 Dec 2009 15:00:00 EST

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Freezing and Thawing Human Embryonic Stem Cells

Thu, 24 Dec 2009 13:00:00 EST

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Wed, 23 Dec 2009 16:00:00 EST

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Culture and Maintenance of Human Embryonic Stem Cells

Tue, 22 Dec 2009 16:50:00 EST

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Mon, 21 Dec 2009 10:00:00 EST

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

PuraMatrix Encapsulation of Cancer Cells

Thu, 17 Dec 2009 17:00:00 EST

This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel.

Extraction of the EPP Component from the Surface EMG

Wed, 16 Dec 2009 16:05:00 EST

The endplate potential (EPP) component can be extracted from the surface EMG using a digital filter. The extracted EPP shows oscillation with a frequency of about 30 Hz.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

Tue, 15 Dec 2009 17:00:00 EST

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

Mon, 14 Dec 2009 17:00:00 EST

Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Fri, 11 Dec 2009 16:00:00 EST

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

The NanoDrop 2000c Spectrophotometer – Microvolume Protein Concentration Determination (German)

Fri, 11 Dec 2009 02:00:00 EST

The NanoDrop 2000c Spectrophotometer – Microvolume Protein Concentration Determination (Spanish)

Fri, 11 Dec 2009 01:00:00 EST

The NanoDrop 2000c Spectrophotometer – Microvolume Protein Concentration Determination (French)

Fri, 11 Dec 2009 00:00:00 EST

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Thu, 10 Dec 2009 17:10:00 EST

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

The 2009 Lindau Nobel Laureate Meeting: Peter Agre, Chemistry 2003

Wed, 09 Dec 2009 20:45:00 EST

American chemist Peter Agre shared the 2003 Nobel Prize in Chemistry for his discovery of aquaporins, the channel proteins that allow water to cross the cell membrane.

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

Tue, 08 Dec 2009 19:45:00 EST

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

Retro-orbital Injection in Adult Zebrafish

Mon, 07 Dec 2009 18:43:00 EST

Here we show how to do retro-orbital injection in adult zebrafish.

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

Fri, 04 Dec 2009 17:45:00 EST

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Thu, 03 Dec 2009 16:30:00 EST

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.