23 articles published in JoVE

• Biology

  
  Combined Nucleotide and Protein Extractions in Caenorhabditis elegans Joslyn Mills¹, Erin McConnell¹, Joshua A. Leitão¹, Louis R. Lapierre^{1 1}Department of Molecular Biology, Cell Biology and Biochemistry, Brown University Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

• Bioengineering

  
  Custom Engineered Tissue Culture Molds from Laser-etched Masters Nicholas J. Kaiser¹, Fabiola Munarin¹, Kareen L.K. Coulombe^{1 1}Center for Biomedical Engineering,, Brown University Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

• Engineering

  
  The Diffusion of Passive Tracers in Laminar Shear Flow Manuchehr Aminian^1,2, Francesca Bernardi¹, Roberto Camassa¹, Daniel M. Harris^1,3, Richard M. McLaughlin^{1 1}Department of Mathematics, University of North Carolina at Chapel Hill, ²Department of Mathematics, Colorado State University, ³School of Engineering, Brown University A protocol for the study of the diffusion of passive tracers in laminar pressure-driven flow is presented. The procedure is applicable to various capillary pipe geometries.

• Bioengineering

  
  A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules Michael A. Monn¹, Jarod Ferreira¹, Jianzhe Yang¹, Haneesh Kesari^{1 1}School of Engineering, Brown University We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 µN up to 10 N and can therefore accommodate a variety of fiber sizes.

• Immunology and Infection

  
  SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells Collins Cheruiyot¹, Zemplen Pataki¹, Robert Williams¹, Bharat Ramratnam^2,3, Ming Li^{3 1}Brown University, ²COBRE Center for Cancer Research, Lifespan Laboratories, Rhode Island and Miriam Hospitals, ³Division of Infectious Diseases, Department of Medicine, Warren Alpert Medical School, Brown University Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

• Environment

  
  Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NO_x Paul K. Wojtal^1,2, David J. Miller^2,3, Mary O'Connor¹, Sydney C. Clark^2,3, Meredith G. Hastings^{2,3 1}Department of Chemistry, Brown University, ²Institute at Brown for Environment and Society, Brown University, ³Department of Earth, Environmental, and Planetary Sciences, Brown University Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

• Immunology and Infection

  
  Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials M. Heath Farris¹, Kara A. Ford², Richard C. Doyle^{3 1}Department of Advanced Technology, The MITRE Corporation, ²Center for Biomedical Engineering, Brown University, ³The MITRE Corporation Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

• Developmental Biology

  
  Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila Eugenia Villa-Cuesta¹, David M. Rand^{2 1}Biology Department, Adelphi University, ²Department of Ecology and Evolutionary Biology, Brown University Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

• Medicine

  
  Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest Brenda Cordeiro¹, Richard Clements^{1,2 1}Cardiothoracic Surgery Research Center, Brown University, ²Rhode Island Hospital and Alpert Medical School, Brown University The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

• Bioengineering

  
  Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix David A. Stout¹, Jennet Toyjanova², Christian Franck^{3 1}Department of Mechanical and Aerospace Engineering, California State University, Long Beach, ²Ximedica, ³School of Engineering, Brown University Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

• Behavior

  
  Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale Barry M. Lester^1,2, Lynne Andreozzi-Fontaine^1,2, Edward Tronick³, Rosemarie Bigsby^{1,2 1}Center for the Study of Children at Risk, Alpert Medical School, Brown University, ²Women & Infants Hospital of Rhode Island, ³University of Massachusetts, Boston The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

• Behavior

  
  Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze Tara K. Jacobson*¹, Jonathan W. Ho*¹, Brendon W. Kent¹, Fang-Chi Yang¹, Rebecca D. Burwell^{1,2 1}Department of Cognitive, Linguistic & Psychological Sciences, Brown University, ²Department of Neuroscience, Brown University We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

• Neuroscience

  
  Automated High-throughput Behavioral Analyses in Zebrafish Larvae Holly Richendrfer¹, Robbert Créton^{1 1}Department of Molecular Biology, Cell Biology and Biochemistry, Brown University Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

• Bioengineering

  
  Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering Bahar Bilgen^1,2, Danielle Chu³, Robert Stefani¹, Roy K. Aaron^{1,2 1}Department of Orthopaedics, The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, ²Center for Restorative and Regenerative Medicine, VA Medical Center, Providence, RI, ³University of Texas Southwestern Medical Center We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

• Neuroscience

  
  In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons Vania Y. Cao^1,2, Yizhou Ye¹, Surjeet S. Mastwal¹, David M. Lovinger³, Rui M. Costa⁴, Kuan H. Wang^{1 1}Unit on Neural Circuits and Adaptive Behaviors, Genes Cognition and Psychosis Program, National Institute of Mental Health, ²Department of Neuroscience, Brown University - National Institutes of Health Graduate Partnership Program, ³Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, ⁴Champalimaud Neuroscience Programme, Champalimaud Center for the Unknown Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

• Neuroscience

  
  Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex Sofia B. Lizarraga^1,2,3, Kathryn R. Coser^1,2, Mark Sabbagh^1,2, Eric M. Morrow^{1,2,3 1}Department of Molecular, Cellular Biology and Biochemistry, Brown University, ²Institute for Brain Science, Brown University, ³Department of Psychiatry and Human Behavior, Warren Alpert School of Medicine, Brown University To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

• Bioengineering

  
  An Experimental System to Study Mechanotransduction in Fetal Lung Cells Yulian Wang¹, Zheping Huang¹, Pritha S. Nayak¹, Juan Sanchez-Esteban^{1 1}Women & Infants Hospital of Rhode Island, Alpert Medical School of Brown University Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

• Biology

  
  Visualizing RNA Localization in Xenopus Oocytes James A. Gagnon¹, Kimberly L. Mowry^{1 1}Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

• Biology

  
  A Protocol for the Production of KLRG1 Tetramer Stephanie C. Terrizzi¹, Cindy Banh¹, Laurent Brossay^{1 1}Department of Molecular Microbiology and Immunology, Brown University This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

• Biology

  
  A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo Ashly Brown¹, Stephen Brown², Debra Ellisor², Nellwyn Hagan¹, Elizabeth Normand¹, Mark Zervas^{2 1}Department of Neuroscience, Division of Biology and Medicine, Brown University, ²Department of Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Brown University Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

• Biology

  
  A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA Katherine H. Watkins¹, Allan Stewart¹, William G. Fairbrother^{1,2 1}Department of Molecular and Cellular Biology, Brown University, ²Center for Computational Molecular Biology, Brown University Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

• Biology

  
  The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis Mark F Brady¹, Jorge Coronel², Robert H Gilman³, David AJ Moore^{4 1}The Warren Alpert Medical School of Brown University, ²Laboratorio de Investigacion de Enfermedades Infecciosas, Universidad Peruana Cayetano Heredia, ³Department of International Health, Johns Hopkins Bloomberg School of Public Health, ⁴Wellcome Trust Centre for Clinical Tropical Medicine, Imperial College London The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

• Biology

  
  Gene-gun Transfection of Hippocampal Neurons Powrnima Joshi¹, Anna Dunaevsky^{1 1}Dept of Neuroscience, Brown University