Rockefeller University View Institution's Website 18 articles published in JoVE Biochemistry Analysis of β-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy Pradeep K. Singh*1, Hanna E. Berk-Rauch*1, Nadine Soplop2, Kunihiro Uryu2, Sidney Strickland1, Hyung Jin Ahn1 1Patricia and John Rosenwald Laboratory of Neurobiology and Genetics, Rockefeller University, 2Electron Microscopy Resource Center, Rockefeller University Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods. Biology Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue Jingyi Chi1, Audrey Crane1, Zhuhao Wu2,3, Paul Cohen1 1Laboratory of Molecular Metabolism, The Rockefeller University, 2Laboratory of Brain Development and Repair, The Rockefeller University, 3Laboratory of Molecular Genetics, The Rockefeller University Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging. Developmental Biology Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs Ksenia Gnedeva1,2, A. J. Hudspeth3, Neil Segil1,2 1Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine of the University of Southern California, 2Caruso Department of Otolaryngology-Head and Neck Surgery, Keck School of Medicine of the University of Southern California, 3Howard Hughes Medical Institute and Laboratory of Sensory Neuroscience, The Rockefeller University Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery. Biology Pulling Membrane Nanotubes from Giant Unilamellar Vesicles Coline Prévost*1,2,3, Feng-Ching Tsai*1,4, Patricia Bassereau1,4, Mijo Simunovic1,5 1Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 2Department of Genetics and Complex Diseases, T. H. Chan School of Public Health, Harvard Medical School, 3Department of Cell Biology, Harvard Medical School, 4Sorbonne Universités, UPMC University Paris 06, 5Center for Studies in Physics and Biology, The Rockefeller University Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro. Immunology and Infection Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo Eliza Prangley1, Terrence Kumar1, Manish P. Ponda1 1Laboratory of Biochemical Genetics and Metabolism, The Rockefeller University Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen. Behavior Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs Jonathan Chabout1,2, Joshua Jones-Macopson1, Erich D. Jarvis1,2,3 1Department of Neurobiology, Duke University, 2Howard Hughes Medical Institute, 3The Rockefeller University Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs. Neuroscience Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana) Julien B. Azimzadeh1, Joshua D. Salvi1 1Laboratory of Sensory Neuroscience, The Rockefeller University The American bullfrog's (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog's sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle's force-displacement relation and measurement of its unforced motion. Biochemistry Protein Complex Affinity Capture from Cryomilled Mammalian Cells John LaCava1,2, Hua Jiang1, Michael P. Rout1 1Laboratory of Cellular and Structural Biology, The Rockefeller University, 2Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads. Genetics High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii Michal Breker1, Kristi Lieberman1, Frej Tulin2, Frederick R. Cross1 1Laboratory of Cell Cycle Genetics, The Rockefeller University, 2Sainsbury Laboratory, University of Cambridge Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput. Immunology and Infection Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry Danae Schulz1,2, Monica R. Mugnier1, Catherine E. Boothroyd1,3, F. Nina Papavasiliou1 1Laboratory of Lymphocyte Biology, Rockefeller University, 2Department of Biology, Harvey Mudd College, 3Masters School African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations. Neuroscience Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells Nadja Zeltner1, Fabien G. Lafaille2, Faranak Fattahi1, Lorenz Studer1 1Developmental Biology, Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, 2St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented. Immunology and Infection Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors Anne Frentzen*1, Kathrin Hueging*1, Julia Bitzegeio2, Thomas Pietschmann1, Eike Steinmann1 1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, 2Aaron Diamond AIDS Research Center, Laboratory of Retrovirology, The Rockefeller University, NY We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny. Biology Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice Yu Chen1,2, Tambudzai Shamu2, Hui Chen3, Peter Besmer3, Charles L. Sawyers2,4, Ping Chi1,5 1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks. Biology PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins Markus Hafner1, Markus Landthaler2, Lukas Burger3, Mohsen Khorshid3, Jean Hausser4, Philipp Berninger4, Andrea Rothballer1, Manuel Ascano1, Anna-Carina Jungkamp2, Mathias Munschauer2, Alexander Ulrich1, Greg S. Wardle1, Scott Dewell5, Mihaela Zavolan3, Thomas Tuschl1 1Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Rockefeller University, 2Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, 3Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 4Biozentrum der Universität Basel and Swiss Institute of Bioinformatics (SIB), 5Genomics Resource Center, Rockefeller University RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs. Biology Direct Restart of a Replication Fork Stalled by a Head-On RNA Polymerase Richard T. Pomerantz1, Mike O'Donnell1 1Howard Hughes Medical Institute, Rockefeller University The fate of the replisome following a collision with a head-on RNA polymerase (RNAP) is unknown. We find that the replisome stalls upon collision with a head-on RNAP, but resumes elongation after displacing the RNAP from DNA. Mfd promotes replication restart by facilitating displacement of the RNAP after the collision. Neuroscience Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae Maurizio Pellegrino1, Takao Nakagawa1, Leslie B. Vosshall1 1Laboratory of Neurogenetics and Behavior, Rockefeller University Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae). Biology High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae Matthieu Louis1, Silvia Piccinotti1, Leslie B. Vosshall1 1Laboratory of Neurogenetics and Behavior, Rockefeller University In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior. Biology MALDI Sample Preparation: the Ultra Thin Layer Method David Fenyo1, Qingjun Wang1, Jeffrey A. DeGrasse1, Julio C. Padovan1, Martine Cadene1, Brian T. Chait1 1Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).