University of Pittsburgh View Institution's Website 71 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. Neuroscience Measuring Constipation in a Drosophila Model of Parkinson's Disease Maggie J. Sodders1, Ming Shen1, Abby L. Olsen1 1Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh This protocol presents an assay for modeling constipation in an alpha-synuclein based Drosophila model of Parkinson's disease. Behavior Real-Time Void Spot Assay Marianela G. Dalghi1, Nicolas Montalbetti1, Travis B. Wheeler2, Gerard Apodaca1, Marcelo D. Carattino1,2 1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day. Neuroscience Pupillometry to Assess Auditory Sensation in Guinea Pigs Marianny Pernia1,2, Manaswini Kar1,2,4, Pilar Montes-Lourido1,2,6, Srivatsun Sadagopan1,2,3,4,5 1Department of Neurobiology, University of Pittsburgh, 2Center for Neuroscience, University of Pittsburgh, 3Department of Bioengineering, University of Pittsburgh, 4Center for Neural Basis of Cognition, University of Pittsburgh, 5Department of Communication Science and Disorders, University of Pittsburgh, 6Department of Transfer and Innovation, USC University Hospital Complex (CHUS), University of Santiago de Compostela Pupillometry, a simple and non-invasive technique, is proposed as a method to determine hearing-in-noise thresholds in normal hearing animals and animal models of various auditory pathologies. Neuroscience TACI: An ImageJ Plugin for 3D Calcium Imaging Analysis Alisa A. Omelchenko1,2, Hua Bai1, Sibtain Hussain1, Jordan J. Tyrrell1,3, Mason Klein4, Lina Ni1 1School of Neuroscience, Virginia Polytechnic Institute and State University, 2CMU-Pitt Joint Computational Biology, School of Medicine, University of Pittsburgh, 3Eastern Virginia Medical School, 4Department of Physics, University of Miami TrackMate Analysis of Calcium Imaging (TACI) is an open-source ImageJ plugin for 3D calcium imaging analysis that examines motion on the z-axis and identifies the maximum value of each z-stack to represent a cell's intensity at the corresponding time point. It can separate neurons overlapping in the lateral (x/y) direction but on different z-planes. Developmental Biology A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse Carla Guzman-Moreno*1, Peizhao Zhang*2, Olivia R. Phillips*1, Mathias Block*3, Benjamin J. Glennon*1, Meghan Holbrook2, Lauren Weigand2, Cecilia W. Lo1, Jiuann-Huey I. Lin1,4 1Department of Developmental Biology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh, 3Département de Biologie, École Normale Supérieure de Lyon, 4Department of Critical Care Medicine, University of Pittsburgh This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction. Biology The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion Nikolaos L. Dedousis1, Lihong Teng1, Alison B. Kohan1 1Division of Endocrinology and Metabolism, Department of Medicine, School of Medicine, University of Pittsburgh The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients. Medicine Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells Marcelo D. Carattino1,2, Wily G. Ruiz1, Gerard Apodaca1,2 1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh This protocol describes a methodology to assess the function of mechanically activated ion channels in native urothelial cells using the fluorescent Ca2+ sensor GCaMP5G. Medicine Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction Ebin Johny1, Partha Dutta1,2,3 1Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, Department of Medicine, University of Pittsburgh, 2Department of Immunology, University of Pittsburgh, 3Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction. Biology Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease Santana M. Lardo1, Sarah J. Hainer1 1Department of Biological Sciences, University of Pittsburgh CUT&RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&RUN. Neuroscience Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats Matthew T. Rich1,2, Yanhua H. Huang1, Mary M. Torregrossa1 1Department of Psychiatry, University of Pittsburgh, 2Department of Psychiatry, Rutgers University The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking. Cancer Research Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research Giovanna Azzarito1, Marta Ewa Szutkowska1, Annalisa Saltari2, Edwin K. Jackson3, Brigitte Leeners1, Marinella Rosselli1, Raghvendra K. Dubey1,3 1Department of Reproductive Endocrinology, Wagistrasse 14, University of Zurich and University Hospital Zurich, 2Department of Dermatology, Wagistrasse 18, University of Zurich and University Hospital Zurich, 3Department of Pharmacology and Chemical Biology, University of Pittsburgh Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research. Biology Isolation of Human Primary Valve Cells for In vitro Disease Modeling Rolando A. Cuevas1, Claire C. Chu1, William J. Moorhead III1, Ryan Wong1, Ibrahim Sultan2, Cynthia St. Hilaire1,3 1Division of Cardiology, Department of Medicine, and the Pittsburgh Heart, Lung, and Blood Vascular Medicine Institute, University of Pittsburgh, 2Division of Cardiac Surgery, Department of Cardiothoracic Surgery, University of Pittsburgh and Heart and Vascular Institute, University of Pittsburgh Medical Center, 3Department of Bioengineering, University of Pittsburgh This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity. Medicine Blind Endotracheal Intubation in Neonatal Rabbits Sergio Benito1, Stephanie Hadley2, Marta Camprubí-Camprubí3, Joan Sanchez-de-Toledo4,5 1Department of Pediatric Critical Care, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, 2Vanderbilt University School of Medicine, 3Neonatology Department, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, Universidad de Barcelona, 4Department of Pediatric Cardiology, Hospital Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, 5Department of Critical Care Medicine, University of Pittsburgh We describe a technique of endotracheal intubation in newborn rabbits after esophageal catheterization with a gastric tube. Immunology and Infection Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients Lindsay Jackson1, William DePas2, Amanda J. Morris1, Kevin Guttman1, Yvonne C W Yau1,3, Valerie Waters1,4 1Translational Medicine, Hospital for Sick Children, 2Center for Microbial Pathogenesis, Department of Pediatrics, University of Pittsburgh, 3Microbiology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 4Infectious Diseases, Department of Pediatrics, Hospital for Sick Children This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients. 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. Medicine Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction Anna L. Lang1,2, William T. Goldsmith3,4, Regina D. Schnegelberger5,6, Gavin E. Arteel6,7, Juliane I. Beier6,7 1Department of Pharmacology and Toxicology, University of Louisville, 2Hepatobiology and Toxicology Program, University of Louisville, 3Department of Physiology and Pharmacology, West Virginia University, 4Center for Inhalation Toxicology, West Virginia University, 5Department of Pharmacology and Chemical Biology, University of Pittsburgh, 6Pittsburgh Liver Research Center, University of Pittsburgh, 7Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Pittsburgh The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe. Cancer Research Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury Hamza O. Yazdani1, Samer Tohme1 1Department of Surgery, University of Pittsburgh We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth. 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. Chemistry A Rhodopsin Transport Assay by High-Content Imaging Analysis Bing Feng1, Xujie Liu1, Yuanyuan Chen1,2 1Department of Ophthalmology, University of Pittsburgh, 2McGowan Institute for Regenerative Medicine, University of Pittsburgh Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell. Biology Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face Anne M. Burrows1,2, Kailey M Omstead1, Ashley R Deutsch3,5, Justin T. Gladman4, Adam Hartstone-Rose3 1Department of Physical Therapy, Duquesne University, 2Department of Anthropology, University of Pittsburgh, 3College of Sciences, North Carolina State University, 4Shared Materials Instrumentation Facility, Duke University, 5Department of Anthropology, University of Florida Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects. Medicine An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney Brittney M. Rush*1, Sarah A Small*1, Donna B. Stolz2, Roderick J. Tan1 1Division of Renal-Electrolyte, Department of Medicine, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time. Bioengineering Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications Ming-Cheng Chen*1, John R. Lake*1, Keith C. Heyde2, Warren C. Ruder1,2 1Department of Bioengineering, University of Pittsburgh, 2Department of Mechanical Engineering, Carnegie Mellon University Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research. Environment Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity Jack E. Watson1, Tyson Robb2, Danielle Andrews-Brown3, Melissa Miller1 1Department of Ecosystem Science and Management, Pennsylvania State University, 2Department of Geography and Environmental Sustainability, State University of New York, Oneonta, 3Department of Geology and Environmental Science, University of Pittsburgh Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site. Immunology and Infection Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics Hyunryul Ryu1, Kyungyong Choi1,2, Yanyan Qu3, Taehong Kwon1,2, Janet S. Lee3,5, Jongyoon Han1,2,4 1Research Laboratory of Electronics, Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 3Department of Medicine, University of Pittsburgh, 4Department of Biological Engineering, Massachusetts Institute of Technology, 5Vascular Medicine Institute, University of Pittsburgh In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases. Immunology and Infection Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis Laura A. Brannelly1,2, Alexandra A. Roberts1, Lee F. Skerratt1, Lee Berger1 1One Health Research Group, College of Public Health, Medical and Veterinary Sciences, James Cook University, 2Department of Biological Sciences, University of Pittsburgh We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis. Medicine Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion Despoina Aslanoglou1, Emily W. George1, Zachary Freyberg1,2 1Department of Psychiatry, University of Pittsburgh, 2Department of Cell Biology, University of Pittsburgh Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells. 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. Cancer Research Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization Frank J. Jenkins1,2, Charles M. Kerr2, Elise Fouquerel2,3, Dana H. Bovbjerg2,4, Patricia L. Opresko2,3 1Departments of Pathology and Infectious Diseases and Microbiology, University of Pittsburgh, 2University of Pittsburgh Cancer Institute, 3Department of Environmental and Occupational Health, University of Pittsburgh, 4Departments of Psychiatry, Psychology, Behavioral & Community Health Sciences, University of Pittsburgh In this article, a detailed protocol for quantifying telomere length using a modified terminal restriction fragment analysis is discussed that provides fast and efficient direct measurement of telomere length. This technique can be applied to a variety of cell sources of DNA for quantifying telomere length. 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. Medicine Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring Mitchell Dyer1, Shannon Haldeman1, Andres Gutierrez1, Lauryn Kohut1, Anirban Sen Gupta2, Matthew D. Neal1 1Department of Surgery, University of Pittsburgh, 2Department of Biomedical Engineering, Case Western Reserve University Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model. Biochemistry Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT) Christina D. King1, Joseph D. Dudenhoeffer1, Liqing Gu2, Adam R. Evans3, Renã A. S. Robinson1 1Department of Chemistry, University of Pittsburgh, 2SGS North America Inc., 3Large Molecule Analytical Development, Pharmaceutical Development & Manufacturing Science, Janssen Research and Development Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls. Bioengineering Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials Keith C. Heyde*1,2, Felicia Y. Scott*3, Sung-Ho Paek3, Ruihua Zhang3, Warren C. Ruder3,4 1Department of Mechanical Engineering, Carnegie Mellon University, 2Engineering Science and Mechanics Program, Virginia Polytechnic Institute and State University, 3Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 4Department of Bioengineering, University of Pittsburgh This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces. Biochemistry Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins Jenna K. Zalewski1, Simone Heber1,2, Joshua H. Mo1, Keith O'Conor1, Jeffrey D. Hildebrand1, Andrew P. VanDemark1 1Department of Biological Sciences, University of Pittsburgh, 2Institute of Structural Biology, German Research Center for Environmental Health We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques. Developmental Biology Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue James E. Baily1, William C.W. Chen2,3, Nusrat Khan4, Iain R. Murray4, Zaniah N. González Galofre4, Johnny Huard5,6, Bruno Péault4,7 1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium. Biology FRET Imaging in Three-dimensional Hydrogels Amalie E. Donius*1, Sylvain V. Bougoin*1,3, Juan M. Taboas1,2 1Department of Oral Biology, Center for Craniofacial Regeneration, McGowan Institute of Regenerative Medicine, University of Pittsburgh, 2Department of Bioengineering, Center for Craniofacial Regeneration, McGowan Institute of Regenerative Medicine, University of Pittsburgh, 3Laerdal AS Förster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described. Biology Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 Karthika Natarajan1,2, Yi Xie1,3, Takeo Nakanishi4, Rebecca S. Moreci5,6, Pancharatnam Jeyasuria7, Arif Hussain1,3,8,9, Douglas D. Ross1,3,8,9,10,11 1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Pharmaceutical Sciences, University of Maryland School of Pharmacy, 3Baltimore VA Medical Center, 4Membrane Transport and Biopharmaceutics, School of Pharmaceutical Sciences, Kanazawa University, 5Obstetrics, Gynecology and Reproductive Science, University of Pittsburgh, 6 With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays. Developmental Biology Quantification of Colonic Stem Cell Mutations Ryan D. Whetstone1, Barry Gold1 1Department of Pharmaceutical Sciences, University of Pittsburgh We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents. 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 Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration Tae-Young Choi1, Mehwish Khaliq1, Sungjin Ko1, Juhoon So1, Donghun Shin1 1Department of Developmental Biology, McGowan Institute for Regenerative Medicine, University of Pittsburgh To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration. Medicine In vitro Functional Characterization of Mouse Colorectal Afferent Endings Bin Feng1, G.F. Gebhart1 1Center for Pain Research, Department of Anesthesiology, University of Pittsburgh This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. Biology Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle William C.W. Chen1, Arman Saparov2,3, Mirko Corselli4, Mihaela Crisan5, Bo Zheng6, Bruno Péault7,8, Johnny Huard9 1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7 Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia. Engineering Writing and Low-Temperature Characterization of Oxide Nanostructures Akash Levy1, Feng Bi1, Mengchen Huang1, Shicheng Lu1, Michelle Tomczyk1, Guanglei Cheng1, Patrick Irvin1, Jeremy Levy1 1Department of Physics, University of Pittsburgh Oxide nanostructures provide new opportunities for science and technology. The interfacial conductivity between LaAlO3 and SrTiO3 can be controlled with near-atomic precision using a conductive atomic force microscopy technique. The protocol for creating and measuring conductive nanostructures at LaAlO3/SrTiO3 interfaces is demonstrated. Medicine A Standardized Obstacle Course for Assessment of Visual Function in Ultra Low Vision and Artificial Vision Amy Catherine Nau1, Christine Pintar1, Christopher Fisher1, Jong-Hyeon Jeong2, KwonHo Jeong2 1UPMC Eye Center, University of Pittsburgh, 2Department of Biostatistics, University of Pittsburgh We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. The course is relatively inexpensive, simple to administer, and has been shown to be reliable and reproducible. 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. Biology Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia Richard Francis1, Cecilia Lo1 1Department of Developmental Biology, University of Pittsburgh An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load. Biology Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection Abrahim I. Orabi1, Kamaldeen A. Muili1, Dong Wang1, Shunqian Jin1, George Perides2, Sohail Z. Husain1 1Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Pittsburgh of UPMC, 2Department of Surgery, Tufts University Medical Center We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided. Engineering Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers Yong Zhao1, Yifan Tang1, Alexander Star1 1Department of Chemistry, University of Pittsburgh We discussed the synthesis of individual graphitic nanocups using a series of techniques including chemical vapor deposition, acid oxidation and probe-tip sonication. By citrate reduction of HAuCl4, the graphitic nanocups were effectively corked with gold nanoparticles due to the chemically reactive edges of the cups. Immunology and Infection PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP Hongshuai Li1,2, Bingyun Li1,3,4 1Department of Orthopaedics, School of Medicine, West Virginia University, 2Department of Orthopaedics, Stem Cell Research Center, University of Pittsburgh, 3WVNano Initiative, 4Mary Babb Randolph Cancer Center Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro. Chemistry Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes Laura S. Kocsis1, Erica Benedetti1, Kay M. Brummond1 1Department of Chemistry, University of Pittsburgh Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions. Bioengineering Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion Nathaniel T. Remlinger1,2, Peter D. Wearden1,3, Thomas W. Gilbert1,2,3,4 1McGowan Institute for Regenerative Medicine, 2Department of Bioengineering, University of Pittsburgh, 3Department of Cardiothoracic Surgery, Children's Hospital of Pittsburgh of UPMC, 4Department of Surgery, University of Pittsburgh A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications. Neuroscience Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging Jeffrey S. Phillips1,2, Adam S. Greenberg1,3, John A. Pyles1,3, Sudhir K. Pathak1,4, Marlene Behrmann1,3, Walter Schneider1,3, Michael J. Tarr1,3 1Center for the Neural Basis of Cognition, 2Department of Psychology, University of Pittsburgh, 3Department of Psychology, Carnegie Mellon University, 4Department of Bioengineering, University of Pittsburgh We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks. Immunology and Infection Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy Peter A. Keyel1, Michelle E. Heid1, Simon C. Watkins2, Russell D. Salter1 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. Biology Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons David Svilar1,2, Conchita Vens3, Robert W. Sobol1,2,4 1Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, 2Hillman Cancer Center, University of Pittsburgh Cancer Institute, 3Department of Experimental Therapy, The Netherlands Cancer Institute, 4Department of Human Genetics, University of Pittsburgh School of Public Health We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins. Medicine A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation Fabrisia Ambrosio1,2,3, G. Kelley Fitzgerald2, Ricardo Ferrari1,2, Giovanna Distefano1,2, George Carvell2 1Department of Physical Medicine and Rehabilitation, University of Pittsburgh, 2Department of Physical Therapy, University of Pittsburgh, 3McGowan Institute for Regenerative Medicine, University of Pittsburgh A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice. Biology Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach Maria Jaramillo1, Ipsita Banerjee1,2 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. Immunology and Infection Growth of Mycobacterium tuberculosis Biofilms Kathleen Kulka1, Graham Hatfull2, Anil K. Ojha1 1Department of Infectious Diseases and Microbiology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis. Immunology and Infection Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays Helene Mens1, Mary Kearney1, Ann Wiegand1, Jonathan Spindler1, Frank Maldarelli1, John W. Mellors2, John M. Coffin3 1The virology Core at the HIV Drug Resistance Program, NCI-Frederick, 2Division of Infectious Diseases, University of Pittsburgh, 3Department of Molecular Biology and Microbiology, Tuffts University Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads. Immunology and Infection Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction Xin Meng1, Gongpu Zhao1, Peijun Zhang1 1Department of Structural Biology, University of Pittsburgh School of Medicine This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method. Medicine Fixed Volume or Fixed Pressure: A Murine Model of Hemorrhagic Shock Lauryn K. Kohut1, Sophie S. Darwiche1, John M. Brumfield1, Alicia M. Frank1, Timothy R. Billiar1 1Department of Surgery, University of Pittsburgh The Hemorrhagic Shock model has been a reliable and reproducible resource facilitating the identification and understanding of signaling cascades associated with inflammation and end-organ damage after trauma. This article provides a step-by-step description of surgical and mechanical aspects associated with the Hemorrhagic Shock experimental procedure in mice. Medicine Pseudofracture: An Acute Peripheral Tissue Trauma Model Sophie S. Darwiche1, Philipp Kobbe2, Roman Pfeifer2, Lauryn Kohut1, Hans-Christoph Pape2, Timothy Billiar1 1Department of Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Aachen Medical Center Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma. Bioengineering Elastomeric PGS Scaffolds in Arterial Tissue Engineering Kee-Won Lee1, Yadong Wang1,2 1Department of Bioengineering, University of Pittsburgh, 2McGowan Institute for Regenerative Medicine, University of Pittsburgh Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period. Medicine Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface Hilaire C. Lam1,2, Augustine M.K. Choi1, Stefan W. Ryter1 1Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Cellular and Molecular Pathology, School of Medicine, University of Pittsburgh Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin. Biology The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker Yue Zhang*1, Luv Kashyap*2, Annabel A. Ferguson2, Alfred L. Fisher2 1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker. Biology Generation of Transgenic C. elegans by Biolistic Transformation Daniel Hochbaum1, Annabel A. Ferguson1, Alfred L. Fisher1 1Department of Medicine, University of Pittsburgh Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals. Biology Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury Chiara Cianciolo Cosentino1, Beth L. Roman2, Iain A. Drummond3, Neil A. Hukriede1 1Department of Developmental Biology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh, 3Department of Medicine and Genetics, Harvard Medical School We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage. Biology Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos Jian Zou1, Xiangyun Wei1,2 1Department of Ophthalmology, University of Pittsburgh, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis. Immunology and Infection Orthotopic Hind-Limb Transplantation in Rats Robert Sucher*1, Rupert Oberhuber*1, Christian Margreiter1, Guido Rumberg1, Rishi Jindal2, WP Andrew Lee2, Raimund Margreiter1, Johann Pratschke1, Stefan Schneeberger1, Gerald Brandacher1 1Department of Visceral, Transplant, and Thoracic Surgery, Daniel Swarovski Research Laboratory, Innsbruck Medical University, 2Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Pittsburgh Medical Center Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research. Biology Isolating Stem Cells from Soft Musculoskeletal Tissues Yong Li1,2,3,4, Haiying Pan1, Johnny Huard1,2,3,4,5 1Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC, 2Department of Bioengineering, University of Pittsburgh, 3Department of Orthopedic Surgery, University of Pittsburgh, 4Department of Pathology, University of Pittsburgh, 5Department of Molecular Genetics & Biochemistry, University of Pittsburgh Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask. Biology Development of automated imaging and analysis for zebrafish chemical screens. Andreas Vogt1, Hiba Codore2, Billy W. Day3,4, Neil A. Hukriede2, Michael Tsang2 1Pharmacology and Chemical Biology, University of Pittsburgh Drug Discovery Institute, 2Department of Microbiology and Molecular Genetics, University of Pittsburgh, 3Department of Pharmaceutical Sciences, University of Pittsburgh, 4Department of Chemistry, University of Pittsburgh We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens. Biology Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis Sagar D. Joshi1, Lance A. Davidson1,2 1Bioengineering, University of Pittsburgh, 2Developmental Biology, University of Pittsburgh Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.
