Temple University View Institution's Website 18 articles published in JoVE Bioengineering A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation Qingliang Yang1, Jordan C. Langston2, Yuan Tang3, Balabhaskar Prabhakarpandian4, Laurie E. Kilpatrick5, Mohammad F. Kiani1,2,6 1Department of Mechanical Engineering, Temple University, 2Department of Bioengineering, Temple University, 3Department of Bioengineering, University of Toledo, 4Biomedical Technology, CFD Research Corporation, 5Center for Inflammation and Lung Research, Department of Microbiology, Immunology and Inflammation, Lewis Katz School of Medicine, Temple University, 6Department of Radiation Oncology, Lewis Katz School of Medicine, Temple University In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease. Neuroscience Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery Rupert D. Smit1, Thomas J. Campion III1, Rachel L. Stingel1, Pushti H. Shah1, Jie Chen1, George M. Smith1 1Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord. Biology Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model Louisiane Perrin1, Theodore Tucker1, Bojana Gligorijevic1,2 1Bioengineering Department, Temple University, 2Cancer Biology Program, Fox Chase Cancer Center Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion. Neuroscience Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord Kathleen M. Keefe1, Ian P. Junker1, Imran S. Sheikh1, Thomas J. Campion1, George M. Smith1 1Shriner's Pediatric Research Center, Temple University This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications. Biology Application of High-speed Super-resolution SPEED Microscopy in Live Primary Cilium Andrew Ruba1, Wangxi Luo1, Weidong Yang1 1Department of Biology, Temple University Recently we mapped the three-dimensional (3D) spatial locations of transport routes for various proteins translocating inside primary cilia in live cells. Here this paper details the experimental setup, the process of biological samples and the data analyses for the 3D super-resolution fluorescence imaging approach newly applied in live primary cilia. Neuroscience Touchscreen Sustained Attention Task (SAT) for Rats Debra A. Bangasser1,2, Brittany Wicks1, David E. Waxler1, Samantha R. Eck1 1Psychology Department, Temple University, 2Neuroscience Program, Temple University Sustained attention, or continuously monitoring situations for intermittent and unpredictable events, is a critical aspect of cognition. Here we detail how to test sustained attention in rats using touchscreen operant chambers. We demonstrate comparable performance in male and female rats, making this task useful for studying attention in both sexes. Chemistry A Protocol for Safe Lithiation Reactions Using Organolithium Reagents Michael R. Gau1, Michael J. Zdilla1 1Department of Chemistry, Temple University The safe and proper use of organolithium reagents is described. Medicine A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy Danielle N. Feather-Schussler1,2, Tanya S. Ferguson1,2 1Department of Medical Genetics and Molecular Biochemistry, Lewiz Katz School of Medicine at Temple University, 2Shriners Hospitals Pediatric Research Center Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups. Behavior Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task Barbara Sargent1, Hendrik Reimann2, Masayoshi Kubo3, Linda Fetters1 1Division of Biokinesiology & Physical Therapy at the Herman Ostrow School of Dentistry, University of Southern California, 2Department of Kinesiology, Temple University, 3Department of Physical Therapy, Niigata University of Health and Welfare A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. Neuroscience Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey Antón Barreiro-Iglesias1, Guixin Zhang2, Michael E. Selzer2,3, Michael I. Shifman2 1Centre for Neuroregeneration, School of Biomedical Sciences, University of Edinburgh, 2Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine, 3Department of Neurology, Temple University School of Medicine Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization. Neuroscience Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection Larra W. Yuelling1, Fang Du1, Peng Li1, Renata E. Muradimova1, Zeng-jie Yang1 1Cancer Biology Program, Fox Chase Cancer Center, Temple University Health System Nestin-expressing progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies. Biology Gene Trapping Using Gal4 in Zebrafish Jorune Balciuniene1, Darius Balciunas1 1Department of Biology, Temple University This protocol describes the method of gene trap insertional mutagenesis using Gal4-VP16 as the primary reporter and GFP/RFP as secondary reporters in zebrafish. Approximately one in ten high-expressing F0 fish yield gene trap progeny co-expressing GFP and RFP. The screening procedure can be readily scaled to adapt to the size of the laboratory performing the insertional mutagenesis screen. Biology Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology Conrad T. Pfeiffer1, Christian E. Schafmeister1 1College of Science and Technology, Temple University The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described. Neuroscience Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis Andrew R. Bauder1, Toby A. Ferguson1 1Center for Neural Repair and Rehabilitation, Temple University In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush. Biology Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS Karthik Mallilankaraman1, Rajesh Kumar Gandhirajan1, Brian J. Hawkins2, Muniswamy Madesh1 1Department of Biochemistry, Temple University, 2Department of Anesthesiology and Pain Medicine, University of Washington An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model. Neuroscience Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice Megan Wright1, Amy Kim2, Young-Jin Son3 1Biology Department, Arcadia University, 2Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs. Neuroscience Live Imaging of Dorsal Root Axons after Rhizotomy Andrew Skuba1, B. Timothy Himes2,3, Young-Jin Son4 1Temple University, Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, 2Medical Research Service, Department of Veterans Affairs Hospital, 3Department of Neurobiology and Anatomy, Drexel University College of Medicine, 4Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS. Medicine Cecal Ligation Puncture Procedure Miguel G. Toscano1, Doina Ganea1, Ana M. Gamero2 1Department of Microbiology and Immunology School of Medicine, Temple University, 2Department of Biochemistry, School of Medicine, Temple University The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.