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
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
JoVE Immunology and Infection
1Division of Pediatrics, MD Anderson Cancer Center - University of Texas
Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.
Published February 2, 2011. Keywords: Immunology, Natural Killer Cells, Tumor Immunology, Antigen Presenting Cells, Cytotoxicity
1Institute of Human Physiology and Clinical Experimental Research, Semmelweis University
We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.
Published November 5, 2011. Keywords: Medicine, ischemia/reperfusion model, stem cell transplantation, confocal microscopy, flow cytometry
1School of Engineering, University of California, Merced
For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.
Published October 3, 2014. Keywords: Bioengineering, Cell Delivery, Matrices, Tissue Engineering, Cardiac Patch, Cell Sheet Engineering
1Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, 2The Beatson Institute for Cancer Research
Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.
Published December 7, 2011. Keywords: Medicine, cancer, cell invasion, imaging, retroviral labeling, RNAi, 3D, Matrix, Matrigel, ECM
1Laboratoire des Multimatériaux et Interfaces, UMR CNRS 5615, Université Lyon1, 2UFR d'Odontologie, Université Lyon1; Service de Consultations et de Traitements Dentaires, Hospices Civils de Lyon, 3UFR d'Odontologie, Université Paris Diderot; Service d'Odontologie, APHP, Hôpital Rothschild
The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.
Published November 9, 2014. Keywords: Medicine, In vitro biocompatibility, dental composites, Live/Deadstaining, 3D imaging, Confocal Microscopy, Time lapse imaging
JoVE Immunology and Infection
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.
Published October 1, 2012. Keywords: Immunology, Cellular Biology, Physiology, streptolysin O, pore-forming toxin, cholesterol-dependent cytolysin, live cell imaging, fluorescence microscopy
1School of Biological and Health Systems Engineering, Arizona State University
The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).
Published September 13, 2012. Keywords: Bioengineering, Genetics, Molecular Biology, Biomedical Engineering, siRNA, transfection, electroporation, microelectrode array, MEA
1Department of Biomedical Engineering, University of Rochester, 2Department of Chemical Engineering, University of Rochester, 3Center for Musculoskeletal Research, University of Rochester Medical Center
This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.
Published October 29, 2013. Keywords: Chemistry, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
1Biomatériaux et Bioingénieriee, INSERM, 2Service Oto-Rhino-Laryngologie, Hôpitaux Universitaires de Strasbourg, 3Faculté de Chirurgie Dentaire, Université de Strasbourg
In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.
Published July 1, 2013. Keywords: Biomedical Engineering, Bioengineering, Medicine, Anatomy, Physiology, Biophysics, Cellular Biology, Molecular Biology, Materials Science, Biomedical and Dental Materials, Composite Materials, Metals and Metallic Materials, Engineering (General), Titanium, pore gradient, implant, in vivo, blood analysis, freeze-extraction, foams, implants, transplantation, clinical applications
1BD Biosciences, Discovery Labware
This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.
Published July 20, 2009. Keywords: Cellular Biology, Tumor Invasion Assay, Chemotaxis, Calcein-AM, Matrigel, Falcon, Fluoroblok, Migration, Invasion, Tumor, BD, Matrigel, Boyden chamber, Motility, Haptotaxis
1Department of Biomedical Engineering, Tufts University
We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.
Published September 19, 2011. Keywords: Bioengineering, fibrin, scaffold, hydrogel, cardiac tissue engineering, contraction force, neonatal cardiomyocytes
JoVE Immunology and Infection
1Department of Hematology and Oncology, University Medical Center Göttingen, 2Microbial Interface Biology Group, Research Center Borstel
We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.
Published September 9, 2014. Keywords: Immunology, macrophages, monocytes, isolation, PBMCs, density gradient, differentiation, Teflon-coated cell culture bags
1Fluxion Biosciences, Inc.
The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.
Published October 27, 2009. Keywords: Medicine, thrombus formation, anti-thrombotic, microfluidic, whole blood assay, IC50, drug screening, platelet, adhesion
1Biomedical Engineering, Tulane University
Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.
Published February 11, 2011. Keywords: Bioengineering, Micropatterning, Photopolymerization, Hydrogels, Cell Culture, Tissue Engineering, Neural Engineering
1Department of Bioengineering, University of Pennsylvania, 2Department of Bioengineering, University of Pennsylvania-School of Medicine
We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.
Published October 26, 2009. Keywords: Cellular Biology, Hydrogel, Tissue Engineering, Biomaterials, Encapsulation, Scaffolds, Bioengineering, Cell Culture, Polymers
1School of Biomedical Engineering and Science, Virginia Tech, 2Department of Engineering Science and Mechanics, Virginia Tech
Contactless dielectrophoresis (cDEP) achieves sorting and enrichment of particles via their intrinsic dielectric properties. Fluidic electrode channels replace metallic electrodes traditional to DEP, suiting cDEP to non-damaging sterile characterization and sorting of biological particles. We demonstrate how to prepare a cDEP microdevice and conduct cell characterization and sorting experiments.
Published September 3, 2013. Keywords: Biomedical Engineering, Medicine, Cellular Biology, Molecular Biology, Bioengineering, Anatomy, Physiology, Biophysics, Physics, Microfluidics, Cell Separation, Microfluidic Analytical Techniques, Electrophoresis, Microchip, cancer diagnosis, cell enrichment, cell sorting, microfluidics, dielectrophoresis, Lab on a chip, cells, imaging
1Department of Biomedical Engineering, Purdue School of Engineering and Technology, Indiana University - Purdue University at Indianapolis
The following protocol provides techniques for encapsulating pancreatic β-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.
Published December 6, 2012. Keywords: Biomedical Engineering, Bioengineering, Tissue Engineering, Cellular Biology, Molecular Biology, Biomaterials, beta cells, β-cell, PEG, PEG-peptide hydrogels, hydrogel, MIN6, poylmers, peptides, spheroids, pancreas
1Antibody Development Department, R&D Systems, Inc., 2Stem Cells and Developmental Biology Department, R&D Systems, Inc.
A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.
Published August 27, 2013. Keywords: Molecular Biology, Cellular Biology, Biochemistry, Biomedical Engineering, Stem Cell Biology, Cancer Biology, Medicine, Bioengineering, Anatomy, Physiology, biology (general), Pathological Conditions, Signs and Symptoms, Wounds and Injuries, Neoplasms, Diagnosis, Therapeutics, Epithelial to mesenchymal transition, EMT, cancer, metastasis, cancer stem cell, cell, assay, immunohistochemistry
1Department of Microbiology and Immunology and Department of Pathology, Queen's University, 2Ask Science Products Inc.
This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.
Published October 18, 2014. Keywords: Molecular Biology, Electroporation, Indium-Tin oxide, signal transduction, gap junctional communication, peptides, Stat3
1Department of Regenerative Medicine, United States Army Institute of Surgical Research
A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.
Published June 1, 2012. Keywords: Bioengineering, Biomedical Engineering, Tissue Engineering, chitosan, microspheres, collagen, hydrogel, cell delivery, adipose-derived stem cells, ASC, CSM
1Laboratory of Neurodevelopement and Repair, University of Science and Technology of China
We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.
Published May 3, 2014. Keywords: Neuroscience, Adult Zebrafish, Retinal Ganglion Cell, Retrograde Labeling, DiI
1ARTORG Center for Biomedical Engineering, University of Bern
This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.
Published February 2, 2012. Keywords: Bioengineering, Intervertebral Disc, Organ Culture, Cartilaginous Endplates, Growth Plate, Cell Viability, Diffusion, Nutrition, Tissue Engineering, Mechanical Loading, Bioreactor
1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's, Harvard Medical School, 2Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 3Brigham and Women's Hospital, Harvard Medical School, 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital
Published October 1, 2007. Keywords: Cellular Biology, tissue engineering, microfluidics, ejection, imaging, bioengineering