Mount Sinai View Institution's Website 13 articles published in JoVE Immunology and Infection Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells Melissa V. Fernandez1, Elizabeth A. Miller2, Nina Bhardwaj3 1Department of Pathology, New York University School of Medicine, 2Division of Infectious Diseases, Department of Medicine, Mount Sinai Medical Center, 3Division of Hematology and Oncology, Hess Center for Science and Medicine, Mount Sinai Medical Center Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1β expression. Biology The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions Yo Suzuki1, Jason Stam1, Mark Novotny2, Nozomu Yachie3, Roger S. Lasken2, Frederick P. Roth3,4 1Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 2Department of Microbial and Environmental Genomics, J. Craig Venter Institute, 3Donnelly Centre & Department of Molecular Genetics, University of Toronto, 4Lunenfeld Research Institute, Mt Sinai Hospital The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions. Biology Examining BCL-2 Family Function with Large Unilamellar Vesicles James J. Asciolla1, Thibaud T. Renault1, Jerry E. Chipuk1 1Department of Oncological Sciences, Department of Dermatology, The Tisch Cancer Institute, The Graduate School of Biological Sciences, Mount Sinai School of Medicine Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented. Neuroscience Extinction Training During the Reconsolidation Window Prevents Recovery of Fear Daniela Schiller1, Candace M. Raio2, Elizabeth A. Phelps3 1Departments of Psychiatry and Neuroscience, and Friedman Brain Institute, Mt. Sinai School of Medicine, 2Department of Psychology, New York University, 3Department of Psychology and Center for Neural Science, New York University Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory). Neuroscience Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia Bjoern Ch. Ludwar1, Colin G. Evans1,2, Elizabeth C. Cropper1 1Fishberg Department of Neuroscience and Friedman Brain Institute, Mt. Sinai School of Medicine, 2Phase Five Communications Inc. We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes. Immunology and Infection High-throughput Detection Method for Influenza Virus Pawan Kumar1, Allison E. Bartoszek1, Thomas M. Moran2, Jack Gorski3, Sanjib Bhattacharyya4, Jose F. Navidad4, Monica S. Thakar1,5, Subramaniam Malarkannan1,6 1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine, 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin, 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens. Immunology and Infection Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation Robert Bowles*1, Sonali Patil*1, Hanna Pincas1, Stuart C. Sealfon1 1Center for Translational Systems Biology and Department of Neurology, Mount Sinai School of Medicine We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels. Biology Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling Keiji Itoh1, Sergei Y. Sokol1 1Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling. Medicine Gene Transfer for Ischemic Heart Failure in a Preclinical Model Kiyotake Ishikawa1, Dennis Ladage1, Lisa Tilemann1, Kenneth Fish1, Yoshiaki Kawase1, Roger J. Hajjar1 1Cardiovascular Research Center, Mount Sinai School of Medicine A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way. Neuroscience Assessment of Social Interaction Behaviors Oksana Kaidanovich-Beilin1, Tatiana Lipina1, Igor Vukobradovic2, John Roder1,3,4,5, James R. Woodgett1,3 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 2Toronto Centre for Phenogenomics, Mount Sinai Hospital, 3Department of Medical Biophysics, University of Toronto, 4Department of Psychology, University of Toronto, 5Department of Psychiatry, University of Toronto Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results. Neuroscience Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze Prasad R. Shirvalkar1, Mathew L. Shapiro1 1Department of Neuroscience, Friedman Brain Institute, Mount Sinai School of Medicine We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals. Immunology and Infection Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy Benjamin Dale1, Gregory P. McNerney2, Deanna L. Thompson2, Wolfgang Hübner3, Thomas Huser2, Benjamin K. Chen1 1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine, 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments. Immunology and Infection Generation of Recombinant Influenza Virus from Plasmid DNA Luis Martínez-Sobrido1, Adolfo García-Sastre2 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Microbiology and Medicine, and Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.