University of Louisville View Institution's Website 26 articles published in JoVE Chemistry Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy Johann V. Hemmer*1, Padmanabh B. Joshi*1, Andrew J. Wilson1 1Department of Chemistry, University of Louisville The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging. Immunology and Infection Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis Jenna M. Olson1, Jonathan M. Warawa1,2 1Department of Microbiology and Immunology, University of Louisville, 2Center for Predictive Medicine, University of Louisville This protocol presents the construction and use of a Simplified Whole Body Plethysmography apparatus to monitor bacterial respiratory disease progression non-invasively. Developmental Biology 3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens Liang Bao1, Mark P. Running1 1Department of Biology, University of Louisville This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period. Developmental Biology The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach John R. Klem1, Raèden Gray1, C. Ben Lovely1 1Alcohol Research Center, Department of Biochemistry and Molecular Genetics, University of Louisville This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos. Cancer Research Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells Shuhan Meng1,2, Aaron G. Whitt1,2,3, Allison Tu2, John W. Eaton1,2,3, Chi Li1,2,3, Kavitha Yaddanapudi4,5,6 1Department of Pharmacology and Toxicology, University of Louisville, 2Experimental Therapeutics Program, Brown Cancer Center, University of Louisville, 3Department of Medicine, University of Louisville, 4Department of Surgery, University of Louisville, 5Immuno-Oncology Program, Brown Cancer Center, University of Louisville, 6Department of Microbiology and Immunology, University of Louisville This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors from embryonic stem cells. Immunology and Infection Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line Katharina M. Scheurlen1, Dylan L. Snook1, Sarah A. Gardner1, Maurice R. Eichenberger1, Susan Galandiuk1 1Price Institute of Surgical Research, Department of Surgery, University of Louisville M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment. Neuroscience Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds Lisa C. Ohman1, Robin F. Krimm1 1Anatomical Sciences and Neurobiology, University of Louisville This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla. Bioengineering Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells Connor S. Centner1, Emily M. Murphy1, Bryce F. Stamp2, Mariah C. Priddy1, John T. Moore1, Paula J. Bates2, Michael A. Menze3, Kavitha Yaddanapudi2, Jonathan A. Kopechek1 1Department of Bioengineering, University of Louisville, 2School of Medicine, University of Louisville, 3Department of Biology, University of Louisville This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents. Bioengineering Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair Takeichiro Nakane1,2,6, Mosha Abulaiti1,2, Yuko Sasaki1, William J. Kowalski3, Bradley B. Keller4,5,7, Hidetoshi Masumoto1,2 1Clinical Translational Research Program, RIKEN Center for Biosystems Dynamics Research, 2Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 3Laboratory of Stem Cell and Neuro-Vascular Biology, Cell and Developmental Biology Center, National Institutes of Health, 4Kosair Charities Pediatric Heart Research Program, Cardiovascular Innovation Institute, University of Louisville, 5Department of Pediatrics, School of Medicine, University of Louisville, 6Department of Cardiovascular Surgery, Mitsubishi Kyoto Hospital, 7 The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases. Medicine Slicing and Culturing Pig Hearts under Physiological Conditions Qinghui Ou*1, Riham R.E. Abouleisa*1, Xian-Liang Tang1, Hamzah R. Juhardeen1, Moustafa H. Meki2, Jessica M. Miller2, Guruprasad Giridharan2, Ayman El-Baz2, Roberto Bolli1, Tamer M.A. Mohamed1,3,4,5,6 1Institute of Molecular Cardiology, Department of Medicine, University of Louisville, 2Department of Bioengineering, University of Louisville, 3Diabetes and Obesity Center, Department of Medicine, University of Louisville, 4Department of Pharmacology and Toxicology, University of Louisville, 5Institute of Cardiovascular Sciences, University of Manchester, 6Faculty of Pharmacy, Zagazig University This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model. Developmental Biology Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography C. Ben Lovely1 1Department of Biochemistry and Molecular Genetics, Alcohol Research Center, University of Louisville This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis. 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. Medicine Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats Natia Q. Kelm1, Jason E. Beare1,2, Amanda J. LeBlanc1,3 1Cardiovascular Innovation Institute, University of Louisville, 2Kentucky Spinal Cord Injury Research Center, University of Louisville, 3Department of Physiology, University of Louisville Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease. Medicine Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research Yoram Fleissig1, Robert M. Reed2,3, Jason E. Beare2,3, Amanda J. LeBlanc2,4, Stuart K. Williams2,4, Christina L. Kaufman1, James B. Hoying2,4 1Christine M. Kleinert Institute for Hand and Microsurgery, 2Cardiovascular Innovation Institute, University of Louisville, 3Kentucky Spinal Cord Injury Research Center, University of Louisville, 4Department of Physiology, University of Louisville Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence. Neuroscience Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats Jason H. Gumbel1, Casey J. Steadman1, Robert F. Hoey1, James E. Armstrong1, Jason D. Fell1, Cui Bo Yang1, Lynnette R. Montgomery1, Charles H. Hubscher1,2 1Department of Anatomical Sciences and Neurobiology, University of Louisville, 2Kentucky Spinal Cord Injury Rehabilitation Center, University of Louisville This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol. Bioengineering Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism Robert A. Skolik1, Mary E. Konkle2, Michael A. Menze1 1Department of Biological Sciences, University of Louisville, 2Department of Chemistry, Ball State University This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow. Immunology and Infection Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death Yuwen Zhang1, Jiaqing Hao1, Yanwen Sun1, Bing Li1 1Department of Microbiology and Immunology, University of Louisville We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels. Bioengineering Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections Hung R. Vuong*1, Kevin M. Tyo*2,3, Jill M. Steinbach-Rankins2,3,4,5 1Department of Chemistry, University of Louisville, 2Department of Pharmacology and Toxicology, University of Louisville, 3Center for Predictive Medicine, University of Louisville, 4Department of Microbiology and Immunology, University of Louisville, 5Department of Bioengineering, University of Louisville This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described. Medicine A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice Xiangbing Wu*1, Yi Ping Zhang*2, Wenrui Qu1, Lisa B. E. Shields2, Christopher B. Shields2,3, Xiao-Ming Xu1 1Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Department of Neurological Surgery and Goodman and Campbell Brain and Spine, Indiana University School of Medicine, 2Norton Neuroscience Institute, Norton Healthcare, 3Department of Anatomical Sciences and Neurobiology, University of Louisville We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice. Engineering Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System Hanwen Yuan1, Scott D. Cambron1, Robert S. Keynton1 1Bioengineering Department, University of Louisville Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and “web-like” structures generated via automated direct writing procedure by means of a 3-axis dispensing system. Neuroscience An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time Starlyn L. M. Okada1, Nicole S. Stivers1, Peter K. Stys2, David P. Stirling1 1KY Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, 2Hotchkiss Brain Institute, Department of Clinical Neurosciences, University of Calgary We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability. Medicine Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury Sevda C. Aslan1, Manpreet K. Chopra1, William B. McKay2, Rodney J. Folz3, Alexander V. Ovechkin1 1Department of Neurological Surgery, University of Louisville, 2Spinal Cord Injury Laboratory, Shepherd Center, 3Department of Medicine: Division of Pulmonary, Critical Care and Sleep Disorders, University of Louisville The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis. Biology Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay Nazarul Hasan1, David Humphrey1, Krista Riggs1, Chuan Hu1 1Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase. Medicine A Simplified Technique for Producing an Ischemic Wound Model Sufan Chien1, Bradon J. Wilhelmi1 1Department of Surgery, University of Louisville We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals. Immunology and Infection Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin Venkatakrishna R. Jala1, Bodduluri Haribabu1 1Microbiology and Immunology, James Graham Brown Cancer Center, University of Louisville This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques. Biology Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells William N. White1, Palaniappan Sethu2 1Department of Mechanical Engineering, University of Louisville, 2Department of Bioengineering, University of Louisville An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.