JoVE Neuroscience
Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window
1Bioengineering, University of California, San Diego, 2La Jolla Bioengineering Institute
Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.
JoVE Immunology and Infection
Isolation of Brain-infiltrating Leukocytes
Department of Neurology, Mayo Clinic College of Medicine
A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.
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 Bioengineering
Micro-particle Image Velocimetry for Velocity Profile Measurements of Micro Blood Flows
1Department of Chemical and Biological Engineering, University of Ottawa, 2Department of Mechanical Engineering, University of Ottawa
Micro-particle image velocimetry (μPIV) is used to visualize paired images of micro particles seeded in blood flows which are cross-correlated to give an accurate velocity profile. Shear rate, maximum velocity, velocity profile shape, and flow rate, each of which has clinical applications, can be derived from these measurements.
JoVE Immunology and Infection
Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure
Vascular Pharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Monash University
This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.
JoVE Immunology and Infection
Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow
1Department of poverty related diseases, Barcelona Centre for International Health Research, 2Confocal Microscopy Unit, University of Barcelona- Scientific and Technological Centers, 3Institució Catalana de Recerca i Estudis Avançats (ICREA)
We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.
JoVE Immunology and Infection
PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP
1Department of Orthopaedics, School of Medicine, West Virginia University, 2Department of Orthopaedics, Stem Cell Research Center, University of Pittsburgh, 3WVNano Initiative, 4Mary Babb Randolph Cancer Center
Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.
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 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
Flow Cytometry Analysis of Immune Cells Within Murine Aortas
1Deptartment of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 2Division of Inflammation Biology, LaJolla Institute for Allergy and Immunology
This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.
JoVE Clinical and Translational Medicine
Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)
Department of Molecular Medicine, University of South Florida Morsani College of Medicine
This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.
JoVE General
Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator
Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.
JoVE General
Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification
1Department of Biochemistry, Microbiology and Immunology, Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3CIHR Program in Neurodegenerative Lipidomics, University of Ottawa, 4Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism
Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.
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 General
Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium
1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School
This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.
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 Neuroscience
High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode
1Center for Neural Science , Korea Institute of Science and Technology (KIST), 2Department of Neuroscience, University of Science and Technology, 3Fab Service Department, Korea Advanced Nano Fab Center
In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.
JoVE General
Cell Electrofusion Visualized with Fluorescence Microscopy
Faculty of Electrical Engineering, University of Ljubljana
In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.
JoVE General
Isolating Stem Cells from Soft Musculoskeletal Tissues
1Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC, 2Department of Bioengineering, University of Pittsburgh, 3Department of Orthopedic Surgery, University of Pittsburgh, 4Department of Pathology, University of Pittsburgh, 5Department of Molecular Genetics & Biochemistry, University of Pittsburgh
Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.
JoVE Clinical and Translational Medicine
Mouse Models for Graft Arteriosclerosis
1Department of Surgery, Yale University School of Medicine, 2Department of Pathology, Yale University School of Medicine
We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.
JoVE Immunology and Infection
Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients
The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.
JoVE Clinical and Translational Medicine
Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model
1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln
An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.
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 Clinical and Translational Medicine
Movement Retraining using Real-time Feedback of Performance
Department of Physical Therapy, University of British Columbia
Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.
JoVE Immunology and Infection
Competitive Homing Assays to Study Gut-tropic T Cell Migration
Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School
Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.
JoVE Immunology and Infection
Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory
This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.
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 Clinical and Translational Medicine
Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System
Department of Laboratory Medicine, Section for Transfusion Medicine, Örebro University Hospital
This article describes the process used by Örebro University Hospital to produce double dose buffy coat platelet concentrates prepared from whole blood donations and treated with the INTERCEPT Blood System for pathogen inactivation. The in vitro quality of the final platelet units are evaluated over 7 days of storage.
JoVE Immunology and Infection
Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions
Department of Physiology and Pharmacology, University of Calgary
This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.
JoVE Immunology and Infection
Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes
1St Vincent’s Institute, Department of Medicine, The University of Melbourne, 2Department of Microbiology and Immunology, The University of Melbourne
Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.
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 General
Isolation of Immune Cells from Primary Tumors
1Tumor Immunity and Tolerance Section, Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, National Cancer Institute - Frederick, 2KEWB Productions
In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.
JoVE Bioengineering
Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy
Department of Biomedical Engineering, University of Rochester
The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.
JoVE Immunology and Infection
Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis
Department of Microbiology and Immunology, University of Rochester
The frog Xenopus laevis provides an attractive alternative non-mammalian model for exploring the ability of heat shock protein such as gp96 to promote antigen-specific CD8 T cell responses. We present methods to study in vivo facilitation of cross-presentation of skin and tumor antigens by gp96.
JoVE General
Determining heat and mechanical pain threshold in inflamed skin of human subjects
Department of Anesthesia, Stanford University School of Medicine
Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.
JoVE Immunology and Infection
Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center
In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.
JoVE General
Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)
1Department of Cancer Biology, Vanderbilt University, 2Departments of Medicine and Cancer Biology, Vanderbilt University
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.
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 General
Microdissection of Zebrafish Embryonic Eye Tissues
Department of Biological Sciences, Purdue University
This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.
JoVE Bioengineering
Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry
We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.
JoVE General
Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
1Department of Biological Sciences, University of Alberta, 2Department of Agriculture, Food and Nutrition Sciences, University of Alberta
An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.
JoVE Immunology and Infection
Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish
1Institute for Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Institute for Applied Informatics, Karlsruhe Institute of Technology, Karlsruhe, Germany
Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.
JoVE Immunology and Infection
Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis
1Department of Kinesiology, Recreation, and Sport, Western Kentucky University, 2Department of Health and Human Performance, University of Houston
Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.
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 General
Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria
Department of Molecular and Cell Biology, University of Connecticut
This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.
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 General
Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging
This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.
JoVE General
Ambulatory ECG Recording in Mice
1Department of Molecular Physiology and Biophysics, Baylor College of Medicine (BCM), 2The Margaret M. and Albert B. Alkek Department of Medicine, Baylor College of Medicine (BCM)
Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.
JoVE Immunology and Infection
Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant
Center for Cell and Gene Therapy, Baylor College of Medicine
A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.
JoVE Neuroscience
The Mouse Forced Swim Test
1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 4The Program in Neuroscience, University of Maryland
The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.
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