Wayne State University View Institution's Website 25 articles published in JoVE Biochemistry An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation Shreenidhi Rajkumar1, Danielle Dixon1, Andrew M. Lipchik2, Joshua J. Gruber1 1Departments of Internal Medicine and the Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 2Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity. Developmental Biology Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo Chenjun Shi1, Chenchen Handler2, Haden Florn1, Jitao Zhang1 1Department of Biomedical Engineering, College of Engineering, Wayne State University, 2Fischell Department of Bioengineering, University of Maryland This protocol was developed to longitudinally monitor the mechanical properties of neural plate tissue during chick embryo neurulation. It is based on the integration of a Brillouin microscope and an on-stage incubation system, enabling live mechanical imaging of neural plate tissue in ex ovo cultured chick embryos. Medicine Mechanical Control of Relaxation Using Intact Cardiac Trabeculae Melissa J. Bukowski1, Benjamin Cavanaugh2, Anita Abbo1, Charles S. Chung1 1Department of Physiology, Wayne State University, 2School of Medicine, Michigan State University-Macomb and Department of Physiology, Wayne State University Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation. Neuroscience Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage Morium Begam1, Neha Narayan1, Drew Mankowski1, Robert Camaj1, Nicholas Murphy1, Kevin Roseni1, Marie E. Pepin1, Jacob M. Blackmer1, Takako I. Jones2, Joseph A. Roche1 1Physical Therapy Program, Department of Health Care Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 2Department of Pharmacology, University of Nevada, Reno School of Medicine The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice. Biology Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint Thomas Wilson1, Navdeep Kaur1, Jason Davis2, Shabana Amanda Ali1,3 1Bone and Joint Center, Henry Ford Health System, 2Department of Orthopedic Surgery, Henry Ford Health System, 3Center for Molecular Medicine and Genetics, Wayne State University Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis. Immunology and Infection Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy Subodh Kumar Samrat1, Haidong Gu1 1Department of Biological Sciences, Wayne State University ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies. Biochemistry Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts Ye Guan1,2, Shiyuan Wang1, Minjun Jin2, Haotian Xu3, Qiong Yang1,4 1Department of Biophysics, University of Michigan, Ann Arbor, 2Department of Chemistry, University of Michigan, Ann Arbor, 3Department of Computer Science, Wayne State University, 4Department of Physics, University of Michigan, Ann Arbor We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions. Medicine Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis Joseph A Roche1, Morium Begam1, Sujay S Galen1 1Department of Health Care Sciences, Physical Therapy Program, College of Pharmacy and Health Sciences, Wayne State University We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle. Biochemistry Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples Tyler Rhinesmith1, Bryan A. Killinger2, Akhil Sharma3, Anna Moszczynska3 1Physiology, Michigan State University, 2Center for Neurodegenerative Science Van Andel Institute, 3Pharmaceutical Sciences, Wayne State University A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented. Bioengineering Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method Cameron B. Pinnock1, Zhengfan Xu1, Mai T. Lam1,2 1Department of Biomedical Engineering, Wayne State University, 2Cardiovascular Research Institute, Wayne State University Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size. Medicine Video Movement Analysis Using Smartphones (ViMAS): A Pilot Study Monica J. Finkbiner1, Kira M. Gaina1, Marie C. McRandall1, Megan M. Wolf1, Vicky M. Pardo1, Kristina Reid1, Brian Adams2, Sujay S. Galen1 1Physical Therapy Program, Department of Healthcare Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, 2Adams Sports Medicine and Physical Therapy This manuscript describes the method to test the concurrent validity of kinematic measures recorded by the smartphone application in comparison to a 3D motion capture system in the sagittal plane. This protocol will enable clinicians to set up smartphones for video capture of human movement. Genetics Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach Zuzer Dhoondia1, Ricci Tarockoff1, Nadra Alhusini1, Scott Medler1, Neha Agarwal1, Athar Ansari1 1Department of Biological Sciences, Wayne State University We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes. Biology Isolation and Primary Culture of Mouse Aortic Endothelial Cells Jie-Mei Wang1, Alex F. Chen2, Kezhong Zhang1 1Center for Molecular Medicine and Genetics, Wayne State University, 2Third Xiangya Hospital and the Institute of Vascular Disease and Translational Medicine, Central South University The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology. Medicine Determining The Electromyographic Fatigue Threshold Following a Single Visit Exercise Test Sujay S. Galen1, Darren R. Guffey2, Jared W. Coburn3, Moh H. Malek1 1Physical Therapy Program and Integrative Physiology of Exercise Laboratory, Department of Health Care Sciences, Wayne State University, 2Sports Medicine and Physical Therapy, MEDSPORT, University of Michigan Health System, 3Department of Kinesiology, California State University, Fullerton This protocol describes the electromyographic fatigue threshold which demarcates between nonfatiguing and fatiguing exercise workloads. This information could be used to develop a more individualized training program. Immunology and Infection Isolation of Leukocytes from the Human Maternal-fetal Interface Yi Xu1, Olesya Plazyo1, Roberto Romero1,2,3,4, Sonia S. Hassan1,5, Nardhy Gomez-Lopez1,5,6 1Perinatology Research Branch, NICHD/NIH/DHHS, 2Department of Obstetrics and Gynecology, University of Michigan, 3Department of Epidemiology and Biostatistics, Michigan State University, 4Department of Molecular Obstetrics and Genetics, Wayne State University, 5Department of Obstetrics and Gynecology, Wayne State University School of Medicine, 6Department of Immunology and Microbiology, Wayne State University School of Medicine Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis. Bioengineering Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds Tonya J. Whitehead1, Harini G. Sundararaghavan1 1Biomedical Engineering, Wayne State University This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days. Biology Imaging Plasma Membrane Deformations With pTIRFM Daniel R. Passmore1, Tejeshwar C. Rao1, Andrew R. Peleman1, Arun Anantharam1 1Department of Biological Sciences, Wayne State University Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape. Neuroscience A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish Jennifer L. Thomas1, Ryan Thummel1,2 1Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 2Department of Opthalmology, Wayne State University School of Medicine Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina. Biology In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes Yanning Wu1, Shuo Wang1, Chunying Li1,2,3 1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro. Immunology and Infection Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge Michael K. Shaw1, Xiao-qing Zhao2, Harley Y. Tse2 1Department of Medicine, Section of Cardiology, St. John-Providence Health System, 2Department of Immunology and Microbiology, Wayne State University School of Medicine Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains. Biology In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin David R. Hyde1, Alan R. Godwin2, Ryan Thummel3 1Department of Biological Sciences, Center for Zebrafish Research, University of Notre Dame, 2Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein’s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth. Medicine MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression Mansoureh Sameni1, Arulselvi Anbalagan1, Mary B. Olive1, Kamiar Moin1,2, Raymond R. Mattingly1,2, Bonnie F. Sloane1,2 1Department of Pharmacology, Wayne State University, 2Barbara Ann Karmanos Cancer Institute, Wayne State University We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness. Neuroscience Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry Francis K. Maina*1, Madiha Khalid*1, Aaron K. Apawu1, Tiffany A. Mathews1 1Department of Chemistry, Wayne State University Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices. Biology In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina Ryan Thummel1, Travis J. Bailey2,3, David R. Hyde2,3 1Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine, 2Department of Biological Sciences, University of Notre Dame, 3Center for Zebrafish Research, University of Notre Dame A method to conditionally knockdown a target protein’s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein’s role in the regenerating or intact retina. Biology Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging Derek J. Averill*1, Joel Garcia*1, Buddhima N. Siriwardena-Mahanama*1, Sashiprabha M. Vithanarachchi*1, Matthew J. Allen1 1Department of Chemistry, Wayne State University We demonstrate the metalation, purification, and characterization of lanthanide complexes. The complexes described here can be conjugated to macromolecules to enable tracking of these molecules using magnetic resonance imaging.