Complement System Proteins:
Serum glycoproteins participating in the host defense mechanism of Complement activation that creates the Complement membrane attack complex. Included are glycoproteins in the various pathways of complement activation (Classical complement pathway; Alternative complement pathway; and Lectin complement pathway).
JoVE Immunology and Infection
Depletion of Specific Cell Populations by Complement Depletion
BloodCenter of Wisconsin, Blood Research Institute
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.
JoVE Immunology and Infection
Measuring the 50% Haemolytic Complement (CH50) Activity of Serum
School of Pharmacy and Medical Sciences, University of South Australia
The classical pathway is activated by antibody and culminates in target cell lysis. The CH50 assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH50 of a serum sample, the calculations and interpreting of results.
JoVE General
Derivation of Human Embryonic Stem Cells by Immunosurgery
Department of Molecular and Cell Biology, Harvard
The ability of human embryonic stem cells to self-renew and differentiate into all cell types of the body suggests that they hold great promise for both medical applications and as a research tool for addressing fundamental questions in development and disease. Here, we provide a concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos by immunosurgical isolation of the inner cell mass.
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.
JoVE Immunology and Infection
Granulocyte-dependent Autoantibody-induced Skin Blistering
1Department of Dermatology, University of Freiburg, 2Kepler High School Freiburg, 3Centre for Biological Signalling Studies (BIOSS), University of Freiburg
In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.
JoVE General
Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine
A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.
JoVE Immunology and Infection
Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols
1Centre for Blood Research, University of British Columbia, 2Department of Pathology and Laboratory Medicine, University of British Columbia, 3Canadian Blood Services, University of British Columbia, 4Department of Chemistry, Life Sciences Centre, University of British Columbia
The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.
JoVE Clinical and Translational Medicine
FISH for Pre-implantation Genetic Diagnosis
This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.
JoVE Clinical and Translational Medicine
Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle
Department of Plastic and Hand Surgery, University of Freiburg Medical Centre
Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.
JoVE General
Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine
Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).
JoVE General
Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures
1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School
Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.
JoVE General
Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching
Saban Research Institute, Childrens Hospital Los Angeles
Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.
JoVE Immunology and Infection
An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells
Infectious Disease Research Center, CHUL (CHUQ), Quebec City, Quebec, Canada
We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.
JoVE General
Single Cell Transfection in Chick Embryos
Department of Medical Neurobiology, Hadassah Medical School - Hebrew University
Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.
JoVE General
Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation
Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.
JoVE General
Presynaptically Silent Synapses Studied with Light Microscopy
1Department of Psychiatry, Washington University School of Medicine, 2Department of Anatomy, Washington University School of Medicine, 3Department of Neurobiology, Washington University School of Medicine
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.
JoVE Immunology and Infection
In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells
This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.
JoVE Immunology and Infection
Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry
Institute for Molecular Infection Biology (IMIB), University of Würzburg
Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.
JoVE General
Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells
We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.
JoVE General
Linearization of the Bradford Protein Assay
Department of Biochemistry, Tel Aviv University
The accuracy and sensitivity of protein determination by the rapid and convenient Bradford assay is compromised by intrinsic nonlinearity. We show a simple linearization procedure that greatly increases the accuracy, improves the sensitivity of the assay about 10-fold, and significantly reduces interference by detergents.
JoVE Immunology and Infection
Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection
Lindsley F. Kimball Research Institute, New York Blood Center
This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.
JoVE Immunology and Infection
Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules
Department of Biology, Case Western Reserve University
We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.
JoVE Immunology and Infection
Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes
Department of Internal Medicine B, University Medicine Greifswald
The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.
JoVE Immunology and Infection
A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, 2Liverpool School of Tropical Medicine, 3Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine
This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.
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 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 Immunology and Infection
Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method
1Institute for Memory Impairments and Neurological Disorders, University of California, 2Physical Medicine & Rehabilitation, University of California, 3Anatomy & Neurobiology, University of California, 4Sue and Bill Gross Stem Cell Research Center, University of California, 5Section of Molecular Biology, University of California, 6Reeve-Irvine Research Center, University of California
Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.
JoVE General
Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR
The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University
An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.
JoVE General
Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging
1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología-Universidad Nacional Autónoma de México, 2Math and Sciences Department, Edison State College
Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.
JoVE Clinical and Translational Medicine
Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department
This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.
JoVE Clinical and Translational Medicine
Modified Technique for Coronary Artery Ligation in Mice
Wallenberg Laboratory, Sahlgrenska Academy, University of Gothenburg
The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.
JoVE Immunology and Infection
Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy
Laboratory of Immune Regulation, Instituto Gulbenkian de Ciência
We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of “niches” within the thymus where T cell development occurs.
JoVE General
Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.
JoVE Clinical and Translational Medicine
In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury
1Department of Surgery, Royal Infirmary of Edinburgh, 2Department of Nephrology, Royal Infirmary of Edinburgh
Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.
JoVE General
Principles of Rodent Surgery for the New Surgeon
Charles River, Research Models and Services
Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.
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 General
Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae
Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.
JoVE General
Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
DOE Plant Research Laboratory, Michigan State University
A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.
JoVE Bioengineering
Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon
1UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Université de Lyon, 2Département Biosciences, INSA de Lyon, Université de Lyon, 3INSERM U758, Ecole Normale Supérieure de Lyon, Université de Lyon, 4Laboratoire de Génie Civil et Ingénierie Environnementale, INSA de Lyon, Université de Lyon
The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.
JoVE Neuroscience
Neural Explant Cultures from Xenopus laevis
Department of Cell Biology, Harvard Medical School
Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.
JoVE Application Notes
Pre-Roll Demo (Standard) - ADVERTISEMENT
For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.
JoVE Application Notes
Pre-Roll Demo (Premium) - ADVERTISEMENT
For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.
JoVE General
Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells
Department of Biochemistry, University of Toronto
Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.
JoVE General
Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure
1Department of Radiology, Massachusetts General Hospital, Harvard Medical School, 2Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 3Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, 4Department of Medicine, Massachusetts General Hospital, Harvard Medical School, 5Center for biotechnology and Informatics, The Methodist Hospital Research Institute, 6Department of Radiology, The Methodist Hospital, Weill Cornell Medical College, 7Bejing University of Chinese Medicine, 8Department of Health Technology and Informatics, The Hong Kong Polytechnic University, 9Department of Radiology, Brigham and Women's Hospital, Harvard Medical School
Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.
JoVE General
Assaying the Kinase Activity of LRRK2 in vitro
Department of Molecular Neuroscience, UCL Institute of Neurology
Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.
JoVE Immunology and Infection
Determining the Phagocytic Activity of Clinical Antibody Samples
1Massachusetts General Hospital, Ragon Institute of MGH, MIT, and Harvard, 2Thayer School of Engineering, Dartmouth College
We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors—providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.
JoVE Clinical and Translational Medicine
Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease
1Department of Pharmacology, University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 2Department of Emergency and Intensive Care, ProMedica Sponsored Research
Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.
JoVE Immunology and Infection
Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models
1Department of Medicine, Vanderbilt University School of Medicine, 2Departments of Medicine and Immunology, Roswell Park Cancer Institute, 3Department of Medicine, University at Buffalo School of Medicine
NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.
JoVE Immunology and Infection
Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy
1Department of Immunology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine
Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.
JoVE Bioengineering
High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT
1Department of Molecular Genetics, Weizmann Institute of Science, 2Department of Biological Regulation, Weizmann Institute of Science, 3Department of Chemical Infrastructure, Weizmann Institute of Science
Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).
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