Highly specialized Epithelial cells
that line the Heart
; Blood vessels
; and lymph vessels, forming the Endothelium
. They are polygonal in shape and joined together by Tight junctions
. The tight junctions allow for variable permeability to specific macromolecules that are transported across the endothelial layer.
JoVE Immunology and Infection
1Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam
Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.
Published August 14, 2014. Keywords: Immunology, Leukocytes, Human Umbilical Vein Endothelial Cells (HUVECs), transmigration, VE-cadherin, PECAM-1, endothelium, transcellular, paracellular
1Department of Biology, American University, 2Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH
The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.
Published September 1, 2014. Keywords: Cancer Biology, Angiogenesis, tube formation, fibroblast, endothelial cell, matrix, 3B-11, basement membrane extract, tubulogenesis
1Klinik und Poliklinik für Anästhesiologie, University of Wurzburg
This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.
Published August 29, 2012. Keywords: Immunology, Neuroscience, Blood-brain barrier, in vitro cell culture models, brain, microvascular endothelial cells, immortalization, cEND
1Department of Bioengineering, University of Pittsburgh, 2Department of Chemical Engineering, University of Pittsburgh
The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.
Published March 27, 2012. Keywords: Stem Cell Biology, Human embryonic stem cells, Endothelial cells, Pancreatic differentiation, Co-culture
1Lombardi 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.
Published April 1, 2011. Keywords: Cellular Biology, Invasion, HUVEC, xCELLigence, impedance, real-time, cell-index
1VECT-HORUS SAS, 2Aix-Marseille Université, CNRS, NICN UMR 7259
The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.
Published June 28, 2014. Keywords: Medicine, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
1Medical Pharmacology and Physiology, University of Missouri, 2Dalton Cardiovascular Research Center
We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.
Published November 25, 2013. Keywords: Basic Protocol, endothelial tubes, microcirculation, calcium signaling, resistance vasculature, Confocal microscopy
JoVE Immunology and Infection
1School of Clinical and Experimental Medicine, University of Birmingham, 2College of Medical and Dental Sciences, University of Birmingham, 3School of Immunity and Infection, University of Birmingham
The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.
Published January 7, 2015. Keywords: Immunology, Endothelial cells, leukocytes, mesenchymal stromal cells, mesenchymal stem cells, co-culture, adhesion, inflammation, recruitment, flow based adhesion assay, Ibidi microslide, neutrophil
1Centre for Vascular Research, University of New South Wales, 2School of Medical Sciences, University of New South Wales
Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.
Published February 19, 2015. Keywords: Bioengineering, Cell adhesion, biosensor, live cell imaging, extracellular matrix, fibronectin, mechanobiology, cell signaling, redox signaling, oxidative stress, myeloperoxidase, endothelium
1School of Sport, Health and Exercise Sciences, Bangor University, 2Department of Rheumatology, Dudley Group of Hospitals NHS Trust, Russells Hall Hospital, 3Arthritis Research UK Epidemiology Unit, University of Manchester
The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.
Published February 7, 2015. Keywords: Medicine, Endothelium, Cardiovascular, Flow-mediated dilatation, Carotid intima-media thickness, Atherosclerosis, Nitric oxide, Microvasculature, Laser Doppler Imaging
1Department of Surgery, University of California, San Francisco, 2Department of Surgery, Veterans Affairs Medical Center, San Francisco, 3VipeRx Lab, Veterans Affairs Medical Center, San Francisco
Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.
Published October 22, 2014. Keywords: Medicine, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
1Department of Cellular and Molecular Medicine, Laboratory of Molecular and Cellular Signaling, KU Leuven
Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.
Published July 16, 2013. Keywords: Cellular Biology, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
1Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, 2Center for Cardiovascular and Pulmonary Research, The Heart Center, The Research Institute at Nationwide Children's Hospital, 3Department of Pediatrics, The Ohio State University
The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.
Published August 21, 2014. Keywords: Cellular Biology, Heart valve, Valve Endothelial Cells (VEC), Fluorescence Activated Cell Sorting (FACS), Mouse, Embryo, Adult, GFP.
JoVE Immunology and Infection
1Department of Immunology, Genetics and Pathology, Uppsala University
The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).
Published November 21, 2014. Keywords: Immunology, In vitro human model system, whole blood, endothelial cells, vascular activation, inflammation, blood coagulation
JoVE Immunology and Infection
1NIHR Biomedical Research Unit, Centre for Liver Research, School of Immunity and Infection, University of Birmingham
Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.
Published March 21, 2014. Keywords: Immunology, Leucocyte trafficking, liver, hepatic sinusoidal endothelial cells, peripheral blood lymphocytes, flow adhesion assay
1Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 2Division of Regenerative Medicine, Tulane National Primate Research Center, 3Department of Microbiology and Immunology, Tulane University School of Medicine, 4Department of Pharmacology, Tulane University School of Medicine
Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.
Published December 15, 2013. Keywords: Bioengineering, rhesus macaque, decellularization, recellularization, detergent, matrix, scaffold, large-organ bioreactor, mesenchymal stem cells
1Division of Renal Diseases and Hypertension, University of Colorado, Denver, 2Department of Integrative Physiology, University of Colorado, Boulder
The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.
Published June 16, 2014. Keywords: Medicine, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
1Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School
Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.
Published April 3, 2014. Keywords: Cellular Biology, nanopodia, TM4SF1, endothelial cell, tumor cell, F-actin, immunofluorescence staining, tetraspanin
1Division 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.
Published February 22, 2013. Keywords: Medicine, Cellular Biology, Anatomy, Physiology, Biomedical Engineering, Biophysics, Surgery, Endothelium, Vascular, Inflammation, Pulmonary Circulation, Intravital Microscopy, endothelial surface layer, endothelial, glycocalyx, pulmonary microvasculature, catheter, tracheostomy, venous, catheterization, lung injury, mouse, animal model
1Department of Psychiatry, Harvard Medical School, 2Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience, McLean Hospital
This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.
Published January 23, 2014. Keywords: Neuroscience, Endothelial cell, mouse, isolation, purification, angiogenesis, embryonic forebrain
1Department of Pulmonary Diseases, Institute for Cardiovascular Research, VU University Medical Center, 2Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center
This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.
Published March 28, 2014. Keywords: Bioengineering, ECIS, Impedance Spectroscopy, Resistance, TEER, Endothelial Barrier, Cell Adhesions, Focal Adhesions, Proliferation, Migration, Motility, Wound Healing
1Department of Biomedical Engineering, Cornell University
We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.
Published December 29, 2010. Keywords: Cellular Biology, Endothelial Cell, Side Specific, Isolation, Aortic Heart Valve, Fibrosa, Ventricularis, Enzymatic Digestion
1Department of Ophthalmology, University of Michigan, 2MidWest Eye Banks
Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet's membranes from donor corneoscleral buttons for transplantation2.
Published September 17, 2014. Keywords: Medicine, DMEK, EK, endothelial keratoplasty, Descemet’s membrane endothelial keratoplasty, corneal transplantation, eye bank, donor tissue preparation
1Department of Physiology, Louisiana State University Health Sciences Center
Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.
Published November 18, 2011. Keywords: Cell Biology, Endothelial cells, actin, cytoskeleton, live-cell imaging, GFP, lamellipodia, stress fibers, kymograph analysis
1Department of Ophthalmology, University of Michigan, 2MidWest Eye Banks
Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.
Published June 12, 2012. Keywords: Medicine, Physiology, Cornea, transplantation, DSAEK, DSEK, endothelial keratoplasty, lamellar, graft, Moria, microkeratome, precut, Fuchs dystrophy
1Australian Centre for Blood Diseases, Monash University
Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.
Published November 12, 2013. Keywords: Bioengineering, Blood-brain barrier, model, cell culture, astrocytes, brain endothelial cells, insert, membranes
JoVE Immunology and Infection
1Department of Anesthesia and Perioperative Care, University of California, San Francisco, 2Graduate Program in Biomedical Sciences, University of California, San Francisco
Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.
Published August 23, 2013. Keywords: Immunology, Cellular Biology, Infection, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Endothelium, Vascular, Neutrophils, Inflammation, Inflammation Mediators, Neutrophil, Leukocyte Adhesion, Endothelial cells, assay
1McAllister Heart Institute, University of North Carolina at Chapel Hill
Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.
Published July 20, 2013. Keywords: Cellular Biology, Molecular Biology, Biomedical Engineering, Bioengineering, Medicine, Cardiology, Cells, Cultured, Embryo, Mammalian, Endothelium, Vascular, Heart, primary cells, cell isolation, endothelial cells, cell, cell culture
1Klinik und Poliklinik für Anästhesiologie, Zentrum für operative Medizin der Universität Würzburg, 2Department of Medicinal Chemistry, Faculty of Life Sciences, University of Vienna
In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro TBI model.
Published October 26, 2013. Keywords: Medicine, stretch injury, traumatic brain injury, blood-brain barrier, brain microvascular endothelial cells (cEND)
JoVE Immunology and Infection
1Department 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.
Published August 8, 2012. Keywords: Immunology, Medicine, Physiology, Cellular Biology, HUVEC, ibidi, leukocyte recruitment, neutrophil, flow chamber
1Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, 2Center for Regenerative Therapeutics, Brigham and Women's Hospital, 3Harvard Medical School, Harvard University, 4Harvard Stem Cell Institute, Harvard University, 5Harvard-MIT Division of Health Sciences and Technology, 6Department of Mechanical Engineering, Massachusetts Institute of Technology
This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.
Published October 16, 2013. Keywords: Bioengineering, Microfluidics, Endothelial Cells, Leukocyte Rolling, HL-60 cells, TNF-α, P-selectin, E-selectin
1Department of Neurology, University of Münster, 2Interdisciplinary Center for Clinical Research (IZKF) Münster, 3Institute of Physiology I — Neuropathophysiology I, University of Münster
Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.
Published November 14, 2014. Keywords: Neuroscience, Blood brain barrier, central nervous system, endothelial cells, immune cell trafficking, neuroinflammation, neurodegeneration, neurovascular unit
1Herman B Wells Center for Pediatric Research, Indiana University School of Medicine
Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.
Published April 13, 2012. Keywords: Cellular Biology, Endothelial colony-forming cells (ECFCs), endothelial progenitor cells (EPCs), single cell colony forming assay, post-natal vasculogenesis, cell culture, cloning
1Lane Department of Computer Science and Electrical Engineering, West Virginia University, 2Department of Cell Biology and Neuroscience, University of California at Riverside
A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging and can be applied for microvascular research.
Published October 21, 2013. Keywords: Bioengineering, Bioengineering, Tissue Engineering, Miniaturization, Microtechnology, Microfluidics, Reflow photoresist, PDMS, Perfusion flow, Primary endothelial cells
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3School of Medicine, University of Pennsylvania, 4Department of Medicine, Division of Cardiology, Duke University Medical Center
We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.
Published January 17, 2012. Keywords: Bioengineering, Fluid Shear Stress, Shear Stress, Shear Force, Endothelium, Endothelial Progenitor Cells, Flow Chamber, Laminar Flow, Flow Circuit, Continuous Flow, Cell Adhesion
JoVE Immunology and Infection
1Genetics and Genomic Sciences Graduate Program, University of Alabama at Birmingham, 2Birmingham Veterans Affairs Medical Center, 3Department of Pathology, University of Alabama at Birmingham, 4Department of Biomedical Engineering, University of Alabama at Birmingham, 5Department of Medicine, University of Alabama at Birmingham
A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.
Published July 2, 2014. Keywords: Immunology, neutrophil adhesion, flow chamber, human umbilical vein endothelial cell (HUVEC), purified ligand
1Institute of Genetic Medicine, Newcastle University, 2UCL Institute of Ophthalmology, University College, London
The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.
Published July 9, 2013. Keywords: Developmental Biology, Neurobiology, Neuroscience, Biomedical Engineering, Cellular Biology, Molecular Biology, Medicine, Anatomy, Physiology, Ophthalmology, Angiogenesis Modulating Agents, Growth and Development, Lymphangiogenesis, Angiogenesis, Mouse Neonatal Retina, Immunofluorescent-Staining, confocal microscopy, imaging, animal model
1Institute of Biomaterials and Biomedical Engineering / Department of Chemical Engineering and Applied Chemistry, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto
Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.
Published December 13, 2010. Keywords: Bioengineering, Tissue engineering, micro-tissue, endothelial cells, collagen gels, modules, 3D tissue culture.
1Department 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.
Published July 21, 2012. Keywords: Biomedical Engineering, Bioengineering, Immunology, Molecular Biology, Genetics, endothelial cell, monocyte arrest, microfluidics, shear stress, cytokine, atherosclerosis, inflammation
1Fondazione Banca Degli Occhi del Veneto O.N.L.U.S., 2Telethon Institute for Genetics & Medicine (T.I.G.E.M.)
The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.
Published June 12, 2012. Keywords: Medicine, Physiology, Human cadaver ocular globe, in situ excision, corneal tissue, in situ evisceration, retinal tissue
1Pole of Cell Biology, Université catholique de Louvain & de Duve Institute
This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.
Published June 6, 2014. Keywords: Cellular Biology, Development, cellular biology, thyroid, organ culture, epithelial morphogenesis, immunostaining, imaging, RNA
JoVE Immunology and Infection
1Department of Biomedicine, Immunoregulation, University of Basel and University Hospital Basel
Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.
Published August 19, 2014. Keywords: Immunology, lymph node, lymph node stromal cells, digestion, isolation, enzymes, fibroblastic reticular cell, lymphatic endothelial cell, blood endothelial cell
1Department 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.
Published February 21, 2013. Keywords: Biomedical Engineering, Bioengineering, Cellular Biology, Biophysics, Molecular Biology, Endothelium, Vascular, Membrane Glycoproteins, Receptors, Leukocyte-Adhesion, bioengineering (general), glycocalyx, mechanical properties, atomic force microscopy, ATM, Endothelial cells, leukocytes, cell wall, cell culture, microscopy, imaging
1Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health
We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.
Published May 20, 2011. Keywords: Developmental Biology, Confocal microscopy, whole-mount immunohistochemistry, mouse embryo, blood vessel, lymphatic vessel, vascular patterning, arterial differentiation
1Department of Medicine, Weill Cornell Medical College, 2Department of Urology, Weill Cornell Medical College
Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.
Published May 15, 2014. Keywords: Medicine, E-selectin, Metastasis, Microslides, Circulating tumor cells, PSMA, Prostate cancer, rolling velocity, immunostaining, HUVECs, flow chambers
1Section of Respiratory, Critical Care and Sleep Medicine, Department of Medicine, University of Illinois, 2Institute for Medicine and Engineering, University of Pennsylvania
Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.
Published September 27, 2012. Keywords: Bioengineering, Biophysics, Biomedical Engineering, Medicine, Cellular Biology, Cell stiffness, biomechanics, microaspiration, cell membrane, cytoskeleton
1Fischell Department of Bioengineering, University of Maryland, 2Institute for Bioscience and Biotechnology Research, University of Maryland
Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.
Published October 17, 2013. Keywords: Bioengineering, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
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.
Published December 21, 2011. Keywords: Molecular Biology, Reactive oxygen species, Calcium, paracrine superoxide, endothelial cells, confocal microscopy
1Angiogenesis Laboratory, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye & Ear Infirmary, 2St. Elizabeth's Medical Center
Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.
Published December 30, 2014. Keywords: Bioengineering, Leukocyte recruitment, endothelium, flow chamber, adhesion, velocity, rolling, integrins, ex vivo
JoVE Immunology and Infection
1Department of Biology and Biochemistry, University of Bath
A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.
Published March 21, 2011. Keywords: Infection, Bacterial pathogen, host cell invasion, Staphylococcus aureus, invasin