Tel Aviv University View Institution's Website 28 articles published in JoVE Cancer Research Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs Neta Moskovits1,2, Ella Itzhaki1,2, Nataly Tarasenko1,2, Eva Chausky1,2, Avital Bareket-Samish3, Aleksandr Kaufman1,2, Raisa Meerson1,2, Salomon M. Stemmer1,2,4 1Felsenstein Medical Research Center, 2Davidoff Center, Rabin Medical Center, 3BioInsight Ltd., 4Sackler Faculty of Medicine, Tel Aviv University The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations. Editorial Endless Worms Most Beautiful: Current Methods For Using Nematodes To Study Evolutionary Developmental Biology Chee Kiang Ewe1, Pradeep M. Joshi2, Joel H. Rothman2 1Department of Neurobiology, Tel Aviv University, 2Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara Engineering Automated Delivery of Microfabricated Targets for Intense Laser Irradiation Experiments Yonatan Gershuni1,2, Michal Elkind1,2, Dolev Roitman1,2, Itamar Cohen1,2, Aviad Tsabary1,2, Deep Sarkar1,2, Ishay Pomerantz1,2 1The School of Physics and Astronomy, Tel Aviv University, 2Tel Aviv University Center for Light-Matter Interaction A protocol is presented for automated irradiation of thin gold foils with high intensity laser pulses. The protocol includes a step-by-step description of the micromachining target fabrication process and a detailed guide for how targets are brought to the laser's focus at a rate of 0.2 Hz. Immunology and Infection T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing Lael Werner1,2, Chen Dor3,4, Naomi Salamon1, Meital Nagar3,4, Dror S. Shouval1,2 1Pediatric Gastroenterology Unit, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, 2Sackler Faculty of Medicine, Tel Aviv University, 3Cancer Research Center, Sheba Medical Center, 4Molecular Hematology Laboratory, Sheba Medical Center The current protocol describes a method for DNA isolation from blood samples and intestinal biopsies, generation of TCRβ and IGH PCR libraries for next-generation sequencing, performance of a NGS run and basic data analysis. Genetics In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity Sampath Kumar Loganathan1, Ahmad Malik1,2, Ellen Langille1,2, Chen Luxenburg3, Daniel Schramek1,2 1Centre for Molecular and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 2Department of Molecular Genetics, University of Toronto, 3Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice. Neuroscience Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System Topaz Altman*1, Roy Maimon*1, Ariel Ionescu*1, Tal Gradus Pery1, Eran Perlson1,2 1Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 2Sagol School of Neuroscience, Tel Aviv University Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers. Behavior Applying Incongruent Visual-Tactile Stimuli during Object Transfer with Vibro-Tactile Feedback Jason Friedman1, Eitan Raveh2, Tehilla Weiss2, Sophi Itkin2, Dafna Niv2, Mor Hani2, Sigal Portnoy2 1Department of Physical Therapy, Sackler Faculty of Medicine, Tel Aviv University, 2Department of Occupational Therapy, Sackler Faculty of Medicine, Tel Aviv University We present a protocol to apply incongruent visual-tactile stimuli during an object transfer task. Specifically, during block transfers, performed while the hand is hidden, a virtual presentation of the block shows random occurrences of false block drops. The protocol also describes adding vibrotactile feedback while performing the motor task. Biology Imaging FITC-dextran as a Reporter for Regulated Exocytosis Ofir Klein1, Amit Roded1, Koret Hirschberg2, Mitsunori Fukuda3, Stephen J. Galli4, Ronit Sagi-Eisenberg1 1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, 2Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, 3Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, 4Departments of Pathology and of Microbiology and Immunology and Sean N. Parker Center for Allergy and Asthma Research, School of Medicine, Stanford University Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically. Cancer Research Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes Nadine Santana-Magal*1, Diana Rasoulouniriana*1, Corey Saperia1, Amit Gutwillig1, Peleg Rider1, Edgar G. Engleman2, Yaron Carmi1 1Department of Pathology, Sackler School of Medicine, Tel-Aviv University, 2Department of Pathology, School of Medicine, Stanford University Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation. Engineering Measurements of Waves in a Wind-wave Tank Under Steady and Time-varying Wind Forcing Andrey Zavadsky1, Lev Shemer1 1School of Mechanical Engineering, Faculty of Engineering, Tel-Aviv University This manuscript describes a fully computer-controlled procedure that allows obtaining reliable statistical parameters from experiments of water waves excited by steady and unsteady wind forcing in a small-scale facility. Behavior Using Virtual Reality to Transfer Motor Skill Knowledge from One Hand to Another Ori Ossmy1,2, Roy Mukamel1,2 1Sagol School of Neuroscience, Tel-Aviv University, 2School of Psychological Sciences, Tel-Aviv University We describe a novel virtual-reality based setup which exploits voluntary control of one hand to improve motor-skill performance in the other (non-trained) hand. This is achieved by providing real-time movement-based sensory feedback as if the non-trained hand is moving. This new approach may be used to enhance rehabilitation of patients with unilateral hemiparesis. Immunology and Infection Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli Ilan Edri*1, Michal Goldenberg*1, Michal Lisnyansky2, Roi Strulovich2, Hadas Newman1,3, Anat Loewenstein1,3, Daniel Khananshvili2, Moshe Giladi2,4, Yoni Haitin2 1Sackler Faculty of Medicine, Tel Aviv University, 2Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 3Department of Ophthalmology, Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, 4Tel Aviv Sourasky Medical Center, affiliated to the Sackler Faculty of Medicine, Tel Aviv University A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies. Behavior Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay Shani Leviatan Ben-Arye1, Hai Yu2, Xi Chen2, Vered Padler-Karavani1 1Department of Cell Research and Immunology, Tel Aviv University, 2Department of Chemistry, University of California-Davis A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases. Genetics Generation of Fluorescent Protein Fusions in Candida Species Sara Gonia1, Judith Berman2, Cheryl A. Gale1 1Department of Pediatrics, University of Minnesota, 2Department of Molecular Microbiology and Biotechnology, Tel Aviv University PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis. Developmental Biology Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry Alona Epshtein1, Lina Sakhneny1, Limor Landsman1 1Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis. Bioengineering Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome Uri Rozovski1, Inbal Hazan-Halevy2, George Calin3, David Harris4, Ping Li4, Zhiming Liu4, Michael J. Keating4, Zeev Estrov4 1Division of Hematology, Davidoff Cancer Center, Rabin Medical Center, 2The Center of Nanoscience and Nanotechnology, Tel Aviv University, 3Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, 4Department of Leukemia, The University of Texas MD Anderson Cancer Center Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest. Immunology and Infection RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells Nadejda Sigal1, Anna Pasechnek1, Anat A. Herskovits1 1The Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel Aviv University Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells. Medicine Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model Assaf Dotan1, Israel Kremer1,2, Orly Gal-Or1, Tami Livnat3, Arie Zigler4, Dan Bourla1, Dov Weinberger1,2,3 1Department of Ophthalmology, Rabin Medical Center, Beilinson Campus, 2Sackler Faculty of Medicine, Tel Aviv University, 3Laboratory of Eye Research, Felsenstein Medical Research Center, 4Racah Institute of Physics, The Hebrew University We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy). Engineering Three-dimensional Particle Tracking Velocimetry for Turbulence Applications: Case of a Jet Flow Jin-Tae Kim1, David Kim1, Alex Liberzon2, Leonardo P. Chamorro1,3 1Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 2School of Mechanical Engineering, Tel Aviv University, 3Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign A three-dimensional particle tracking velocimetry (3D-PTV) system based on a high-speed camera with a four-view splitter is described here. The technique is applied to a jet flow from a circular pipe in the vicinity of ten diameters downstream at Reynolds number Re ≈ 7,000. Immunology and Infection Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis Nurit P. Azouz1,2, Mitsunori Fukuda3, Marc E. Rothenberg2, Ronit Sagi-Eisenberg1 1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, 2Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, 3Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University The goal of the present protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time analyses of the secretory granule's morphology. Medicine Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment Hadas Bar-Joseph1,2, Salomon Marcello Stemmer2,3, Ilan Tsarfaty2,4, Ruth Shalgi1,2, Irit Ben-Aharon2,3 1Department of Cell and Developmental Biology, Tel Aviv University, 2Sackler Faculty of Medicine, Tel Aviv University, 3Institute of Oncology, Davidoff Center and Rabin Medical Center, 4Department of Clinical Microbiology and Immunology, Tel Aviv University We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects. Biology Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells Shiran Ferber*1, Galia Tiram*1, Ronit Satchi-Fainaro1 1Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.). Biology Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS) Yoray Sharon1, Lina Alon1, Sarah Glanz1, Charlotte Servais1, Neta Erez1 1Department of Pathology, Sackler School of Medicine, Tel Aviv University Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFRα as a surface marker. Bioengineering A Microfluidic Device for Studying Multiple Distinct Strains Guy Aidelberg*1, Yifat Goldshmidt*1, Iftach Nachman1 1Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography. Biology Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System Amos Danielli1,2, Noga Porat3, Marcelo Ehrlich4, Ady Arie1 1Department of Physical Electronics, Faculty of Engineering, Tel Aviv University, 2Department of Biomedical Engineering, Washington University in St. Louis, 3Department of Biological Sciences, University of Illinois, 4Department of Cell Research and Immunology, Tel Aviv University Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins. Biology Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells Edward Avezov1, Efrat Ron1, Yana Izenshtein1, Yosef Adan1, Gerardo Z. Lederkremer1 1Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC. Biology Linearization of the Bradford Protein Assay Orna Ernst1, Tsaffrir Zor1 1Department of Biochemistry, Tel Aviv University The accuracy and sensitivity of protein determination by the rapid and convenient Bradford assay is compromised by intrinsic nonlinearity. We show a simple linearization procedure that greatly increases the accuracy, improves the sensitivity of the assay about 10-fold, and significantly reduces interference by detergents. Biology Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast Dor Salomon1, Guido Sessa1 1Department of Plant Sciences, Tel Aviv University In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.