41 articles published in JoVE
1Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania
In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.
Published September 29, 2014. Keywords: Cellular Biology, melanocyte-like cells, heart, mouse, atrial myocytes, primary isolation
1Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, 2Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, 3Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, 4Center for Sleep and Circadian Neurobiology, Perelman School of Medicine, University of Pennsylvania
Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.
Published October 16, 2013. Keywords: Medicine, Anatomy, Physiology, Pharmacology, Anesthesia, Inhalation, Behavioral Research, General anesthesia, loss of righting reflex, isoflurane, anesthetic sensitivity, animal model
1Department of Cell and Developmental Biology, University of Pennsylvania, 2Biomedical Graduate Studies, University of Pennsylvania, 3Department of Animal Biology and Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, 4Department of Genetics, University of Pennsylvania, 5Department of Biology, University of Pennsylvania
The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.
Published October 9, 2013. Keywords: Cellular Biology, Developmental Biology, Spermatogenesis, STA-PUT, cell separation, Spermatogenesis, spermatids, spermatocytes, spermatogonia, sperm, velocity sedimentation
1Department of Neurology, Perelman School of Medicine, University of Pennsylvania, 2Center for Cognitive Neuroscience, University of Pennsylvania, 3Veterans Affairs Boston Healthcare System, 4Harold Goodglass Aphasia Research Center, Boston University School of Medicine, 5Department of Neurology, Boston University School of Medicine
We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.
Published July 2, 2013. Keywords: Medicine, Neurobiology, Neuroscience, Anatomy, Physiology, Biomedical Engineering, Molecular Biology, Neurology, Stroke, Aphasia, Transcranial Magnetic Stimulation, TMS, language, neurorehabilitation, optimal site-finding, functional magnetic resonance imaging, fMRI, brain, stimulation, imaging, clinical techniques, clinical applications
1Cancer Prevention and Control Program, Fox Chase Cancer Center, 2Department of Psychiatry, University of Pennsylvania, 3Department of Psychology, Drexel University, 4Department of Medicine, Fox Chase Cancer Center, 5Cancer Prevention and Control Program, The Cancer Institute of New Jersey
This article describes how to conduct minimal erythema dose (MED) testing in order to determine the lowest dose of ultraviolet radiation that will cause erythema (burning) when administered to an individual.
Published May 28, 2013. Keywords: Medicine, Anatomy, Physiology, Dermatology, Analytical, Diagnostic, Therapeutic Techniques, Equipment, Health Care, Minimal erythema dose (MED) testing, skin sensitivity, ultraviolet radiation, spectrophotometry, UV exposure, psoriasis, acne, eczema, clinical techniques
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania
Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.
Published May 20, 2013. Keywords: Biochemistry, Chemistry, Molecular Biology, Cellular Biology, Physiology, Medicine, Pharmacology, Genetics, Genomics, Mass Spectrometry, MS, Metabolism, Metabolomics, untargeted, extraction, lipids, accurate mass, liquid chromatography, ultraperformance liquid chromatography, UPLC, high resolution mass spectrometry, HRMS, spectrometry
1Division of Human Genetics, Children's Hospital of Philadelphia Research Institute, 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania
The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.
Published April 19, 2013. Keywords: Molecular Biology, Genetics, Biochemistry, Microbiology, Medicine, Proteins, DNA-Binding Proteins, Transcription Factors, Chromatin Immunoprecipitation, Genes, chromatin, immunoprecipitation, ChIP, DNA, PCR, sequencing, antibody, cross-link, cell culture, assay
1Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania
This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.
Published April 6, 2013. Keywords: Stem Cell Biology, Medicine, Neuroscience, Neurobiology, Bioengineering, Biomedical Engineering, Molecular Biology, Cellular Biology, Anatomy, Physiology, stem cells, neural crest, hair, human, bulge, flow cytometry, hair follicles, regenerative medicine, iPS cells, isolation, cell culture
1Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, 2Department of Physiology, Perelman School of Medicine, University of Pennsylvania, 3Department of Anatomy and Cell Biology, School of Dental Medicine, School of Dental Medicine, University of Pennsylvania
Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.
Published January 31, 2013. Keywords: Anatomy, Physiology, Cellular Biology, Biophysics, Medicine, Biomedical Engineering, Surgery, Muscles, Muscular Diseases, Animal Experimentation, Chemicals and Drugs, muscular dystrophy, muscle function, muscle damage, muscular dystrophies, mouse, animal model
1Department of Flow and Image Cytometry, Roswell Park Cancer Institute, 2Flow Cytometry & Cell Sorting Resource Laboratory, University of Pennsylvania, 3SciGro, Inc., 4Department of Pathology and Laboratory Medicine, University of Pennsylvania
Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.
Published December 13, 2012. Keywords: Cellular Biology, Molecular Biology, Cell tracking, PKH26, CFSE, membrane dyes, dye dilution, proliferation modeling, lymphocytes
JoVE Immunology and Infection
1Center for Sleep and Circadian Neurobiology, University of Pennsylvania Perelman School of Medicine
To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NFκB.
Published December 4, 2012. Keywords: Immunology, Neuroscience, Medicine, Physiology, Pathology, Microbiology, immune response, sleep, Drosophila, infection, bacteria, luciferase reporter assay, animal model
1Division of Neurology, Children's Hospital of Philadelphia, 2Neuroscience Graduate Group, Perelman School of Medicine at the University of Pennsylvania, 3Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania
A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.
Published November 19, 2012. Keywords: Neuroscience, Medicine, Anatomy, Physiology, hippocampus, traumatic brain injury, electrophysiology, patch clamp, voltage sensitive dye, extracellular recording, high-performance liquid chromatography, gas chromatography-mass spectrometry
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
1Department 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.
Published September 25, 2012. Keywords: Cancer Biology, Medicine, Molecular Biology, glioblastoma multiforme, mouse, brain tumor, bioluminescent imaging, stereotactic rodent surgery
1Division of Gastroenterology, Hepatology & Nutrition, Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Medicine, University of Pennsylvania
A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.
Published June 29, 2012. Keywords: Physiology, Medicine, Liver, fibrosis, portal fibroblast, liver perfusion, myofibroblast, biliary fibrosis
JoVE Immunology and Infection
1Institute of Immunology, Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Pathobiology, School of Veterinary Medicine, University of Pennsylvania
Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.
Published June 5, 2012. Keywords: Immunology, Infection, Microbiology, helminth, parasite, mouse, Th2, lung, inflammation, granuloma, alternative activation, macrophage
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.
Published May 2, 2012. Keywords: Medicine, FDG-PET/CT, atherosclerosis, vascular inflammation, quantitative radiology, imaging
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
1Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, 2Department of Pharmacology, University of Pennsylvania
This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).
Published November 17, 2011. Keywords: Bioengineering, lipids, extraction, stable isotope dilution, chiral chromatography, electron capture, mass spectrometry
1Department of Biochemistry and Biophysics, University of Pennsylvania
Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.
Published September 30, 2011. Keywords: Molecular Biology, Neuroscience, Hsp104, AAA+, disaggregase, heat shock, amyloid, prion
1Department of Biomedical Engineering, Duke University, 2School of Medicine, Duke University, 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania
A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.
Published September 9, 2011. Keywords: Bioengineering, Stent, Titanium, Thrombosis, Endothelial Progenitor Cell, Endothelium, Biomaterial, Biocompatibility, Bioengineering, Translational Medicine, Vascular Surgery, Porcine
1Department of Neurology, University of Connecticut Health Center, 2Department of Neurology, School of Medicine, University of Pennsylvania, 3Department of Neurosurgery, Hartford Hospital, 4Department of Neurosurgery, School of Medicine, University of Pennsylvania
The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.
Published August 24, 2011. Keywords: Neuroscience, stroke, intracerebral hemorrhage, mice, animal model
1Neuroscience Graduate Group, University of Pennsylvania School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine
Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.
Published July 27, 2011. Keywords: Cell Biology, Yeast, plasmid, transformation, PEG/LioAc, high-throughput screen
1Department of Biology, University of Pennsylvania
Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.
Published July 23, 2011. Keywords: Cell biology, oocyte, microinjection, meiosis, meiotic maturation, aneuploidy
1Gene Expression and Regulation, The Wistar Institute, University of Pennsylvania
Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Published July 14, 2011. Keywords: Molecular Biology, Telomerase, protein expression, purification, chromatography, RNA isolation, TRAP
1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California
We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.
Published May 4, 2011. Keywords: Neuroscience, brain, blood, oxygenation, phosphorescence, imaging
1Department of Pharmacology, University of Pennsylvania, 2The Penn Genome Frontiers Institute, University of Pennsylvania
In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.
Published April 25, 2011. Keywords: Neuroscience, single-cell, transcriptome, aRNA amplification, RT-PCR, molecular biology, gene expression
1Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania
Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.
Published February 27, 2011. Keywords: Developmental Biology, Stable isotope, amino acid quantitation, organic acid quantitation, nematodes, metabolism
1Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, Institute of Diabetes Obesity and Metabolism, Institute for Regenerative Medicine, Department of Pediatrics, University of Pennsylvania-School of Medicine
We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.
Published December 7, 2010. Keywords: Immunology, CldU, IDU, Thymidine Analogue, immunofluorescence, Lineage-Tracing, Mouse, Cell Division, Replication, Turnover, Stem Cell
JoVE Immunology and Infection
1Penn Ovarian Cancer Research Center, Center for Research on Reproduction and Womans Health, Department of Obstetrics and Gynecology, University of Pennsylvania-School of Medicine, 2Women's Cancer Program, Fox Chase Cancer Center
To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.
Published November 28, 2010. Keywords: Immunology, Ovarian cancer, syngeneic, orthotopic, katushka (TurboFP635), in vivo imaging, immunocompetent mouse model of ovarian cancer, deep tumors
1Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, 2Department of Internal Medicine and Department of Psychiatry, School of Medicine, Washington University in St. Louis, 3Veterans Affairs Medical Center, 4School of Dental Medicine, Department of Biochemistry, University of Pennsylvania-School of Medicine, 5Monell Chemical Senses Center, 6Monell Chemical Senses Center
Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.
Published September 18, 2010. Keywords: JoVE Medicine, tongue, human, taste cells, fungiform papillae, biopsy
JoVE Immunology and Infection
1Department of Microbiology, Penn Genome Frontiers Institute, University of Pennsylvania
Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.
Published August 25, 2010. Keywords: cellular biology, RNAi, high-throughput screening, virus-host interactions, Drosophila, viral infections
1Department of Pharmacology, University of Pennsylvania
The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.
Published August 10, 2010. Keywords: Cellular Biology, substrata stiffness, polyacrylamide, hydrogel, synthetic matrix, extracellular matrix, ECM
1Department of Cell and Developmental Biology, University of Pennsylvania
We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.
Published July 16, 2010. Keywords: Cellular Biology, zebrafish, transgenesis, microinjection, GFP, enhancers, transposon
1Department of Cell and Developmental Biology, University of Pennsylvania-School of Medicine
This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.
Published March 4, 2010. Keywords: Developmental Biology, adult, zebrafish, organs, dissection, anatomy
1Department of Biochemistry and Biophysics, University of Pennsylvania
We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.
Published March 3, 2010. Keywords: Cellular Biology, oxygen, phosphorescence, porphyrin, dendrimer, imaging, nanosensor, two-photon
1Department of Neuroscience, University of Pennsylvania-School of Medicine, 2Department of Physiology, University of Pennsylvania-School of Medicine
This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.
Published December 4, 2009. Keywords: Cellular Biology, naphthylstyryl-pyridinium dye, di-4-ANEPPDHQ, enteric nervous system, submucous plexus, myenteric plexus, potentiometric probes, enteric plexuses, neural networks, gut, optical recording, voltage-sensitive dyes
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
1Department of Bioengineering, University of Pennsylvania
The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.
Published October 21, 2009. Keywords: Cellular Biology, Electrospinning, Photocrosslinking, Photopatterning, Tissue Engineering, Scaffolds, Biomaterials, Bioengineering
1Institute for Medicine and Engineering, University of Pennsylvania
Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.
Published July 23, 2008. Keywords: immunology, issue 17, blood, neutrophils, neutrophil polymorphonuclear granulocytes, cell separation, cell isolation
1Department of Bioengineering, University of Pennsylvania, 2University of Washington
In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.
Published October 1, 2007. Keywords: Cellular biology, mechanotransduction, traction force, microfabrication