University of Pittsburgh, School of Medicine 30 articles published in JoVE Neuroscience In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion Jeremy Y. Gedeon1,2,3, Jorge Baruch Pineda-Farias2,3, Michael S. Gold2,3 1Center for Neuroscience at the University of Pittsburgh, 2Department of Neurobiology, University of Pittsburgh School of Medicine, 3Pittsburgh Center for Pain Research, University of Pittsburgh Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity. Biology Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids Erin Crawford*1, Heather L. Mentrup*2,3, Elizabeth A. Novak2,3, Kevin P. Mollen2,3 1Division of Pediatric Surgery, UPMC Children’s Hospital of Pittsburgh, 3Department of Surgery, University of Pittsburgh School of Medicine We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer. Bioengineering Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs Meagan J. Makarcyzk1,2, Zhong Alan Li1,3, Ilhan Yu1, Haruyo Yagi1, Xiurui Zhang1, Lauren Yocum1, Eileen Li1, Madalyn R. Fritch1, Qi Gao4, Bruce A. Bunnell5, Stuart B. Goodman4,6, Rocky S. Tuan1,8, Peter G. Alexander1,7, Hang Lin1,2,7 1Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 2Department of Bioengineering, University of Pittsburgh Swanson School of Engineering, 3Department of Neurobiology, University of Pittsburgh School of Medicine, 4Department of Orthopaedic Surgery, Stanford University, 5Department of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, 6Department of Bioengineering, Stanford University, 7McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 8The Chinese University of Hong Kong We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip. Biology Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction Wily G. Ruiz1, Dennis R. Clayton1, Marianela G. Dalghi1, Nicolas Montalbetti1, Marcelo D. Carattino1, Gerard Apodaca1 1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine Methods are described for the generation of large amounts of recombinant adenoviruses, which can then be used to transduce the native rodent urothelium allowing for expression of transgenes or downregulation of endogenous gene products. Medicine In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents Stephanie L. Daugherty1, Keara M. Healy1, Jonathan M. Beckel1 1Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized rodent. Biology Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium Lyndsay L. Leach1, G. Burch Fisher2, Jeffrey M. Gross1,3 1Department of Ophthalmology, Louis J. Fox Center for Vision Restoration, University of Pittsburgh School of Medicine, 2Earth Research Institute, University of California, Santa Barbara, 3Department of Developmental Biology, University of Pittsburgh School of Medicine This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed. Developmental Biology A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells Aneta Przepiorski1, Amanda E. Crunk1, Teresa M. Holm2, Veronika Sander2, Alan J. Davidson2, Neil A. Hukriede1,3 1Department of Developmental Biology, University of Pittsburgh, School of Medicine, 2Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, 3Center for Critical Care Nephrology, University of Pittsburgh, School of Medicine Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches. Biology In vivo Evaluation of Mucociliary Clearance in Mice Kyle S. Feldman1, Maliha Zahid1 1Department of Developmental Biology, University of Pittsburgh School of Medicine In this publication, we describe protocols for assessing airway mucociliary clearance (MCC) in mice in vivo utilizing dual-modality radionuclide imaging. This protocol is designed for a single photon emission computed tomography (SPECT) and computed tomography (CT) acquisition protocol using mouse whole body (MWB) collimators in a dual SPECT/CT system. Biology In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide Kyle S. Feldman1, Maliha Zahid1 1Department of Developmental Biology, University of Pittsburgh School of Medicine We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half. Immunology and Infection Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells Tiao Li1, Chen Long1, Kristen V. Fanning1, Chunbin Zou1 1Acute Lung Injury Center of Excellence, Pulmonary, Allergy, Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells. Biology Measuring RAN Peptide Toxicity in C. elegans Paige Rudich1, Carley Snoznik2, Noah Puleo2, Todd Lamitina1,2 1Graduate Program in Cell Biology and Molecular Physiology, University of Pittsburgh, 2Department of Pediatrics, University of Pittsburgh School of Medicine Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans. Developmental Biology Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells Wei Feng1, Andrew Przysinda1, Guang Li1 1Department of Developmental Biology, University of Pittsburgh School of Medicine Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects. Medicine Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice Sidney Mahan1, Mingjun Liu1, Richard A. Baylis2,3, Delphine Gomez1,4 1Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, 2Robert M. Berne Cardiovascular Research Center, University of Virginia, 3Department of Biochemistry and Molecular Genetics, University of Virginia, 4Division of Cardiology, University of Pittsburgh School of Medicine We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice. Biology Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima Jee-Hyun Um*1,2, Young Yeon Kim*1,2, Toren Finkel3, Jeanho Yun1,2 1Peripheral Neuropathy Research Center, College of Medicine, Dong-A University, 2Department of Biochemistry, College of Medicine, Dong-A University, 3Aging Institute, Department of Medicine, University of Pittsburgh School of Medicine Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry. Biology Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis Shinichiro Yokota1,2, Yoshihiro Ono2, Toshimasa Nakao2, Peng Zhang3, George K. Michalopoulos4,5, Zahida Khan3,4,5,6 1Department of Surgery, Jichi Medical University, 2Department of Surgery, University of Pittsburgh School of Medicine, 3Department of Pediatrics, University of Pittsburgh School of Medicine, 4Department of Pathology, University of Pittsburgh School of Medicine, 5Pittsburgh Liver Research Center, University of Pittsburgh, 6McGowan Institute for Regenerative Medicine, University of Pittsburgh Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents. Biology Primary Cell Cultures from the Mouse Retinal Pigment Epithelium Peng Shang1, Nadezda A. Stepicheva1, Stacey Hose1, J. Samuel Zigler, Jr.2, Debasish Sinha1,2 1Department of Ophthalmology, University of Pittsburgh School of Medicine, 2Wilmer Eye Institute, The Johns Hopkins University School of Medicine The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE. Medicine Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation Wyatt E. Lanik1, Lily Xu2, Cliff J. Luke1, Elise Z. Hu2, Pranjal Agrawal2, Victoria S. Liu2, Rajesh Kumar1, Alexa M. Bolock1, Congrong Ma3, Misty Good1 1Division of Newborn Medicine, Department of Pediatrics, Washington University School of Medicine, 2Washington University, 3Division of Newborn Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice. Immunology and Infection Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates Alexander G. White*1, Pauline Maiello*1, M. Teresa Coleman1, Jaime A. Tomko1, L. James Frye1, Charles A. Scanga1, Philana Ling Lin2, JoAnne L. Flynn1 1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, 2 Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation. Immunology and Infection Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins Jill A. Dembowski1, Neal A. Deluca1 1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins. Neuroscience Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0 Rashed Harun1,2,3, Christine M. Grassi2, Miranda J. Munoz2,4, Amy K. Wagner1,2,3 1Center for Neuroscience, University of Pittsburgh, 2Department of Physical Medicine & Rehabilitation, University of Pittsburgh, School of Medicine, 3Safar Center for Resuscitation Research, University of Pittsburgh, 4Department of Biological Sciences, Mellon College of Science, Carnegie Mellon University Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics. Biochemistry Measuring Protein Binding to F-actin by Co-sedimentation Jonathon A. Heier1, Daniel J. Dickinson2, Adam V. Kwiatkowski1 1Department of Cell Biology, University of Pittsburgh School of Medicine, 2Department of Biology, University of North Carolina at Chapel Hill This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction. Genetics Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project Francis R. G. Amrit1, Arjumand Ghazi1 1Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID. Developmental Biology Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment Destiny L. Baars1, Kendra A. Takle*1,2, Jonathon Heier*1,3, Francisco Pelegri1 1Laboratory of Genetics, University of Wisconsin, 2Department of Neurobiology, University of Massachusetts Medical School, 3Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species. Medicine Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography Ravirajsinh N. Jadeja1, Vikrant Rachakonda2, Zsolt Bagi3, Sandeep Khurana1 1Section of Gastroenterology and Hepatology, Georgia Regents University, 2Division of Gastroenterology and Hepatology, University of Pittsburgh School of Medicine, 3Vascular Biology Center, Georgia Regents University Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter. Medicine Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye Larry Kagemann1,2, Bo Wang2, Gadi Wollstein1, Hiroshi Ishikawa1,2, Brandon Mentley1, Ian Sigal1,2,3, Richard A Bilonick1,4, Joel S Schuman1,2,3 1Department of Ophthalmology, UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, 2Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, 3The McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 4Deptartment of Biostatistics, Graduate School of Public Health, University of Pittsburgh Trabecular meshwork (TM) migration into Schlemm’s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ. Developmental Biology In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow Christopher C. Rymer1,2, Sunder Sims-Lucas1,2 1 This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney. Medicine Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology F. Aura Kullmann1, Stephanie L. Daugherty2, William C. de Groat2, Lori A. Birder1,2 1Department of Medicine, Renal division, University of Pittsburgh School of Medicine, 2Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology, and pathology. Neuroscience Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking Megan L. Brady1, Charles E. Moon1, Tija C. Jacob1 1Pharmacology & Chemical Biology Department, University of Pittsburgh School of Medicine Here we demonstrate the use of fluorescent Alexa dye coupled to α-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds α-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved. Biology The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins Kristine M. Cihil1,2, Agnieszka Swiatecka-Urban1,2 1Department of Nephrology, Children's Hospital of Pittsburgh of UPMC, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins. Bioengineering Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions Sangmi Jun1, Gongpu Zhao1, Jiying Ning1, Gregory A. Gibson2, Simon C. Watkins2, Peijun Zhang1 1Department of Structural Biology, University of Pittsburgh School of Medicine, 2Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.
