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The Rockefeller University

21 ARTICLES PUBLISHED IN JoVE

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Biology

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice
Yu Chen 1,2, Tambudzai Shamu 2, Hui Chen 3, Peter Besmer 3, Charles L. Sawyers 2,4, Ping Chi 1,5
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.

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Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Chun-Chun Chen 1, Kazuhiro Wada 2, Erich D. Jarvis 1
1Howard Hughes Medical Institute, Department of Neurobiology, Duke University , 2Department of Biological Sciences, Hokkaido University

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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Biology

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors
Saranga Naganathan 1, Amy Grunbeck 1, He Tian 1, Thomas Huber 1, Thomas P. Sakmar 1
1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

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Biology

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells
Pierre Dourlen 1,2, Clemence Levet 1, Alexandre Mejat 1, Alexis Gambis 3, Bertrand Mollereau 1
1Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon, 2INSERM U744, Institut Pasteur de Lille, Université Lille-Nord de France, 3Howard Hughes Medical Institute, Laboratory of Apoptosis and Cancer, The Rockefeller University

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

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Neuroscience

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling
Gustavo Arriaga 1, Joshua J. Macopson 1, Erich D. Jarvis 1,2
1Department of Neurobiology, Duke University Medical Center, 2Howard Hughes Medical Institute

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

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Biology

Gavaging Adult Zebrafish
Chereen Collymore 1,2, Skye Rasmussen 2, Ravi J. Tolwani 2
1Tri-Institutional Training Program in Laboratory Animal Medicine and Science Memorial Sloan-Kettering Cancer Center, Weill Cornell Medical College, The Rockefeller University, 2Comparative Bioscience Center, The Rockefeller University

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

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Neuroscience

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells
Nadja Zeltner 1, Fabien G. Lafaille 2, Faranak Fattahi 1, Lorenz Studer 1
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.

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Developmental Biology

Imaging Subcellular Structures in the Living Zebrafish Embryo
Peter Engerer 1, Gabriela Plucinska 1,2, Rachel Thong 1,3, Laura Trovò 1, Dominik Paquet 4,5,6, Leanne Godinho 1
1Institute of Neuronal Cell Biology, Technische Universität München, 2Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3Faculty of Biology, Ludwig-Maximilians-Universität-München, 4Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-Universität-München, 5German Center for Neurodegenerative Diseases, 6Laboratory of Brain Development and Repair, The Rockefeller University

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

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Behavior

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs
Jonathan Chabout 1,2, Joshua Jones-Macopson 1, Erich D. Jarvis 1,2,3
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.

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Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells
John LaCava 1,2, Hua Jiang 1, Michael P. Rout 1
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.

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JoVE Journal

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii
Michal Breker 1, Kristi Lieberman 1, Frej Tulin 2, Frederick R. Cross 1
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.

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Neuroscience

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)
Julien B. Azimzadeh 1, Joshua D. Salvi 1
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.

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Biology

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles
Coline Prévost *1,2,3, Feng-Ching Tsai *1,4, Patricia Bassereau 1,4, Mijo Simunovic 1,5
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.

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Immunology and Infection

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo
Eliza Prangley 1, Terrence Kumar 1, Manish P. Ponda 1
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.

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Developmental Biology

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs
Ksenia Gnedeva 1,2, A. J. Hudspeth 3, Neil Segil 1,2
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.

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Biology

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue
Jingyi Chi 1, Audrey Crane 1, Zhuhao Wu 2,3, Paul Cohen 1
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.

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Genetics

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology
Laetitia Chauve *1, Jérémie Le Pen *2,3,5, Francesca Hodge 1, Pia Todtenhaupt 1, Laura Biggins 1, Eric A. Miska 2,3,4, Simon Andrews 1, Olivia Casanueva 1
1Babraham Institute, 2Gurdon Institute, University of Cambridge, 3Department of Genetics, University of Cambridge, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute, 5Laboratory of Virology and Infectious Disease, The Rockefeller University

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

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Immunology and Infection

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes
Veronica Jové *1, Krithika Venkataraman *1, Thomas M. Gabel 2, Laura B. Duvall 2
1Laboratory of Neurogenetics and Behavior, The Rockefeller University, 2Department of Biological Sciences, Columbia University

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

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Biology

Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory
Ana-Maria Tanasescu 1
1Laboratory of Membrane Biology and Biophysics, The Rockefeller University

This protocol presents essential cell culture techniques and practices to be used in the research cell culture laboratory to avoid contamination by fungi and bacteria. Within the category of bacteria, special emphasis will be placed on preventing mycoplasma contamination.  

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Immunology and Infection

Gene Editing of Primary Rhesus Macaque B Cells
Harald Hartweger 1, Rajeev Gautam 2, Yoshiaki Nishimura 2, Fabian Schmidt 3,5, Kai-Hui Yao 1, Amelia Escolano 1,6, Mila Jankovic 1, Malcolm A. Martin 2, Michel C. Nussenzweig 1,4
1Laboratory of Molecular Immunology, The Rockefeller University, 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Laboratory of Retrovirology, The Rockefeller University, 4Howard Hughes Medical Institute, The Rockefeller University, 5Laboratory of Applied Virology and Precision Medicine, King Abdullah University of Science and Technology (KAUST), 6Vaccine and Immunotherapy Center, Wistar Institute

We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies.

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Biochemistry

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy
Htet Ng *1, Masuda Begum *1, Gabriella N. L. Chua *1,2, Shixin Liu 1
1Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, 2Tri-Institutional PhD Program in Chemical Biology

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

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