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 Clinical and Translational Medicine
Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)
1Division of Cardiovascular Medicine, University of Pennsylvania, Perelman School of Medicine, 2Department of Radiology, University of Pennsylvania, Perelman School of Medicine, 3Department of Dermatology, University of Pennsylvania, Perelman School of Medicine
There is great need to identify atherosclerosis non-invasively, and here we demonstrate how FDG-PET/CT can be used to detect and quantify atherosclerotic plaque activity and vascular inflammation.
JoVE Clinical and Translational Medicine
Cecal Ligation Puncture Procedure
1Department of Microbiology and Immunology School of Medicine, Temple University, 2Department of Biochemistry, School of Medicine, Temple University
The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
JoVE Immunology and Infection
Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice
1Department of Pharmacology, University of Illinois at Chicago, 2Department of Anesthesiology, University of Illinois at Chicago
Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.
JoVE Immunology and Infection
Non-invasive Imaging of Leukocyte Homing and Migration in vivo
1Department of Pathology and Immunology, Washington University in St. Louis, 2National Institute of Neurological Disorders and Stroke, NINDS, NIH - National Institute of Health
Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.
JoVE Immunology and Infection
Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy
Department of Pharmacology, University of Saskatchewan
We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.
JoVE Clinical and Translational Medicine
Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation
Department of Surgery, University of Bonn
Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.
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 Clinical and Translational Medicine
The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice
Pharmazentrum Frankfurt / ZAFES, Goethe University Hospital Frankfurt
Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.
JoVE General
Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS
1Department of Biochemistry, Temple University, 2Department of Anesthesiology and Pain Medicine, University of Washington
An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.
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 Bioengineering
Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring
1Department of Radiology, University of Nebraska Medical Center, 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center
Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.
JoVE General
Investigating Intestinal Inflammation in DSS-induced Model of IBD
Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.
JoVE Immunology and Infection
Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine
1Department of Pediatrics, Emory University, 2Department of Pathology & Laboratory Medicine, Emory University
Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.
JoVE Immunology and Infection
Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine
A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.
JoVE Clinical and Translational Medicine
Pseudofracture: An Acute Peripheral Tissue Trauma Model
1Department of Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Aachen Medical Center
Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.
JoVE Neuroscience
A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain
1Department of Physics, University of California, San Diego, 2Department of Engineering Science and Mechanics, Pennsylvania State University, 3Department of Neurosurgery, Pennsylvania State University, 4Section of Neurobiology, University of California, San Diego
We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.
JoVE Clinical and Translational Medicine
In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis
1Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, 2Institute for Biological and Medical Imaging, Helmholtz Zentrum München und Technische Universität München, 3Department of Electrical and Computer Engineering, Northeastern University
We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.
JoVE General
Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis
1Department of Biomedical Engineering, Tulane University, 2Department of Biomedical Engineering, University of Virginia, 3Center for Stem Cell Research and Regenerative Medicine, Tulane University
This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.
JoVE General
Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts
We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.
JoVE General
Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)
Department of Pathology, Sackler School of Medicine, Tel Aviv University
Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFRα as a surface marker.
JoVE Immunology and Infection
A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry
1Department of Biological Sciences, University of Alberta, 2División de Estudios de Posgrado e Investigación, Facultad de Odontología, Universidad Nacional Autónoma de México, 3Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México
Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei
JoVE Immunology and Infection
Murine Model of Allergen Induced Asthma
Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.
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 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 Clinical and Translational Medicine
A Mouse Model of in Utero Transplantation
1Department of Surgery, University of California, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, 3Biomedical Sciences Program, University of California
The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique
JoVE Clinical and Translational Medicine
Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme
Department of Radiation Oncology, University of Pennsylvania
We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.
JoVE Immunology and Infection
Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies
1Research Group Immune Regulation, Helmholtz Centre for Infection Research, 2Research Group Infection Immunology, Institute of Medical Microbiology, Otto-von-Guericke University, 3Department of Experimental Immunology, Helmholtz Centre for Infection Research
We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.
JoVE Neuroscience
A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation
1Center for Neural Development and Disease, Department of Neurology, Child Neurology Division, University of Rochester, 2Department of Neurobiology and Anatomy, University of Rochester
We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.
JoVE Bioengineering
Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts
Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago
Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.
JoVE Immunology and Infection
Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging
1Experimental and Clinical Research Center, A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, 2Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine
Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.
JoVE General
Protein Transfection of Mouse Lung
Department of Medicine, St. Luke's Roosevelt Medical Center
Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.
JoVE Immunology and Infection
Isolation of Adipose Tissue Immune Cells
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine
Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.
JoVE Clinical and Translational Medicine
Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock
Department of Surgery, University of Pittsburgh
The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice.
JoVE General
Pull-down of Calmodulin-binding Proteins
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.
JoVE General
Measurement of Cellular Chemotaxis with ECIS/Taxis
1Molecular and Cell Biology, University of Connecticut, 2University of Connecticut
The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.
JoVE Immunology and Infection
Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
1Department of Medicine, Vanderbilt University School of Medicine, 2Department of Microbiology and Immunology, Vanderbilt University School of Medicine
Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.
JoVE Clinical and Translational Medicine
Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface
1Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Cellular and Molecular Pathology, School of Medicine, University of Pittsburgh
Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.
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
A Simple Protocol for Extracting Hemocytes from Wild Caterpillars
Department of Biological Sciences, The George Washington University
Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.
JoVE Immunology and Infection
An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response
1Biology Department, The City College of New York, CUNY, 2The Graduate Center, The City University of New York
Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.
JoVE Clinical and Translational Medicine
In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer
Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine
The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.
JoVE Neuroscience
Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine
In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.
JoVE Bioengineering
Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples
1Department of Biomedical Engineering, Cornell University, 2BioCytics, Inc., 3Carolina BioOncology Institute, PLLC
Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.
JoVE General
The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation
1Department of Medical Pharmacology and Physiology, University of Missouri, 2Dalton Cardiovascular Research Center, University of Missouri
A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.
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 Immunology and Infection
Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice
The Walter and Eliza Hall Institute of Medical Research
A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.
JoVE Neuroscience
Assessing Burrowing, Nest Construction, and Hoarding in Mice
Department of Experimental Psychology, University of Oxford
Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute “environmental enrichment” for the mice, and embody the “Refinement” aspect of the “3 Rs”18.
JoVE Immunology and Infection
Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae
Department of Molecular and Biomedical Sciences, University of Maine
The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.
JoVE Neuroscience
Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis
1Research Service, Veterans Administration Medical Center, Memphis, TN, 2Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, 3Department of Anatomy/Neurobiology, University of Tennessee Health Science Center, Memphis, TN
A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.
More Results...
