Cell Wall:
The outermost layer of a cell in most Plants; Bacteria; Fungi; and Algae. The cell wall is usually a rigid structure that lies external to the Cell membrane, and provides a protective barrier against physical or chemical agents.
JoVE General
Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast
Department of Biological Sciences and Purdue Center for Cancer Research, Purdue University
Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.
JoVE General
Glycan Profiling of Plant Cell Wall Polymers using Microarrays
1Australian Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, 2Plant Cell Biology Research Centre, School of Botany, University of Melbourne, 3CSIRO Plant Industry, Black Mountain Laboratories, 4Department of Plant Biology and Biotechnology, University of Copenhagen
A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.
JoVE General
Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin
Department of Biology, Penn State University
We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.
JoVE General
Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates
1Great Lakes Bioenergy Research Center, Michigan State University (MSU), 2Great Lakes Bioenergy Research Center and DOE-Plant Research Lab, Michigan State University (MSU)
Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.
JoVE General
Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin
1Great Lakes Bioenergy Research Center, Michigan State University (MSU), 2Great Lakes Bioenergy Research Center and DOE-Plant Research Lab, Michigan State University (MSU)
Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.
JoVE General
Label-free in situ Imaging of Lignification in Plant Cell Walls
1Energy Biosciences Institute, University of California, Berkeley, 2Molecular Foundry, Lawrence Berkeley National Laboratory, 3Physical Biosciences Division, Lawrence Berkeley National Laboratory
A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.
JoVE Bioengineering
Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy
1AG Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, 2Helmholtz Zentrum München
Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.
JoVE Immunology and Infection
Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection
Center for Bio/Molecular Science and Engineering, Naval Research Laboratory
A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.
JoVE General
High-throughput Purification of Affinity-tagged Recombinant Proteins
Department of Life Sciences, Imperial College London
We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.
JoVE Immunology and Infection
Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
Department of Immunology, University of Toronto
Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.
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
Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells
School of Biosciences, University of Birmingham
The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.
JoVE Immunology and Infection
Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
1Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, 2Laboratory for Infectious Disease Research, University of Missouri-Columbia
Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.
JoVE Clinical and Translational Medicine
A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells
Lombardi Comprehensive Cancer Center, Georgetown University
This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.
JoVE General
Preparation of Drosophila S2 cells for Light Microscopy
Department of Cell Biology and Anatomy, University of Arizona (UOA)
Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.
JoVE General
Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, 2Flow Cytometry & Cell Sorting Resource Laboratory, University of Pennsylvania, 3SciGro, Inc., 4Department of Pathology and Laboratory Medicine, University of Pennsylvania
Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.
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 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
Isolation of Functional Cardiac Immune Cells
Department of Cell Biology and Anatomy, University of South Carolina- School of Medicine
This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.
JoVE Immunology and Infection
Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus
1Department of Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, 2Department of Obstetrics and Gynecology, College of Medicine, University of Arkansas for Medical Sciences, 3Department of Pathology, College of Medicine, University of Arkansas for Medical Sciences
Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.
JoVE Neuroscience
The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow
1Department of Neurosurgery, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco - UCSF, 2Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, 3Department of Neuroscience and Neurology, College of Physicians and Surgeons, Columbia University, 4Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences, 5Center for Motor Neuron Biology and Disease, College of Physicians and Surgeons, Columbia University
The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.
JoVE General
Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation
1Brigham and Women's Hospital / Harvard Medical School, Department of Medicine, Cardiovascular Division, 2Weill Institute for Cell and Molecular Biology & Department of Biomedical Engineering, Cornell University
We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.
JoVE Bioengineering
On-Chip Endothelial Inflammatory Phenotyping
Department of Biomedical Engineering, University of California, Davis
Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.
JoVE Neuroscience
Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay
1 Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 2Department of Neurosurgery, University of Florida
This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.
JoVE Chemistry
Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance
Department of Chemistry, University of Toronto
Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.
JoVE Neuroscience
Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa
This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).
JoVE General
In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion
Department of Biochemistry, University of Nebraska, Lincoln
The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.
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 Neuroscience
Transcriptome Analysis of Single Cells
1Department of Pharmacology, University of Pennsylvania, 2The Penn Genome Frontiers Institute, University of Pennsylvania
In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.
JoVE General
Blood Collection from the American Horseshoe Crab, Limulus Polyphemus
1Department of Molecular and Cell Biology, University of California, Davis, 2Marine Biological Laboratory - MBL- woods hole, 3Department of Biological Sciences, Hunter College of CUNY
The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and α2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.
JoVE Bioengineering
Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments
1School of Dentistry, Cardiff Institute of Tissue Engineering & Repair, Cardiff University, 2Shandong Qianfoshan Hospital, Shandong University School of Medicine, 3Dermatology and Ophthalmology Research, Institute for Regenerative Cures, University of California at Davis
This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).
JoVE General
OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides
1Energy Biosciences Institute, University of California, Berkeley, 2Department of Plant and Microbial Biology, University of California, Berkeley
A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.
JoVE Neuroscience
A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field
1Institute for Biomaterials and Biomedical Engineering, University of Toronto, 2Lyndhurst Centre, Toronto Rehabilitation Institute, 3Department of Surgery, University of Toronto
In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.
JoVE General
In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy
1Department of Physics, University of Wisconsin - Milwaukee, 2Department of Biological Sciences, University of Wisconsin - Milwaukee
By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of Förster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.
JoVE Neuroscience
Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Sue and Bill Gross Stem Cell Center, University of California, Irvine, 3Institute for Immunology, University of California, Irvine
The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.
JoVE Immunology and Infection
Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells
Centre for Immunity, Infection and Evolution, University of Edinburgh
An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.
JoVE Neuroscience
Inducing Dendritic Growth in Cultured Sympathetic Neurons
Department of Molecular Biosciences, University of California, Davis
We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.
JoVE Neuroscience
Preparation of Drosophila Central Neurons for in situ Patch Clamping
School of Life Sciences, Arizona State University
In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.
JoVE General
A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation
1Max Planck Institute of Molecular Plant Physiology, 2University of Kaiserslautern
We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.
JoVE General
Dissection and Staining of Drosophila Larval Ovaries
Department of Biological Regulation, Weizmann Institute of Science
How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.
JoVE Editorial
October 2012: This Month in JoVE
1Department of Ophthalmology, Massachusetts Eye and Ear, 2JoVE Content Production
Here are some highlights from the October 2012 Issue of Journal of Visualized Experiments (JoVE).
JoVE Bioengineering
Microfluidic Device for Recreating a Tumor Microenvironment in Vitro
Department of Chemical Engineering, University Of Massachusetts Amherst
We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.
JoVE Bioengineering
Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening
1Department of Biomedical Engineering, University of Texas at San Antonio, 2Department of Biology, University of Texas at San Antonio
We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.
JoVE Clinical and Translational Medicine
Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University, 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University, 3Omics Laboratory, University of Iowa, 4Department of Ophthalmology and Visual Sciences, University of Iowa
This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.
JoVE Immunology and Infection
Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis
Louisiana State University Health Sciences Center
Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.
JoVE Clinical and Translational Medicine
The α-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva
1Department of Medicine, Weill Cornell Medical College, 2Department of Oral Biology, University of Missouri-Kansas City-School of Dentistry, 3Department of Pharmacology and Toxicology, University of Missouri Kansas City- School of Pharmacy, 4Regional Hospital, Bamenda, NWP, Cameroon, 5Mezam Polyclinic HIV/AIDS Treatment Center, Cameroon, 6Institute for Human Genetics and Biochemistry
A CD4 enumeration method, the α-test, is described which uses whole saliva to provide rapid and accurate CD4 counts. The α-test costs pennies and eliminates the need for technical training, costly reagents such as monoclonal antibodies, instrumentation, refrigeration, transport of samples, as well as collection and handling of blood.
JoVE Clinical and Translational Medicine
Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
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 Neuroscience
Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations
Department of Experimental Neurobiology, The Neurosciences Institute
We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.
JoVE General
Fabrication of the Thermoplastic Microfluidic Channels
Department of Biomedical Engineering, Boston University
Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.
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