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Tel Aviv University

27 ARTICLES PUBLISHED IN JoVE

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Biology

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast
Dor Salomon 1, Guido Sessa 1
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.

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Biology

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells
Edward Avezov 1, Efrat Ron 1, Yana Izenshtein 1, Yosef Adan 1, Gerardo Z. Lederkremer 1
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.

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Biology

Linearization of the Bradford Protein Assay
Orna Ernst 1, Tsaffrir Zor 1
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.

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Biology

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System
Amos Danielli 1,2, Noga Porat 3, Marcelo Ehrlich 4, Ady Arie 1
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.

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Bioengineering

A Microfluidic Device for Studying Multiple Distinct Strains
Guy Aidelberg *1, Yifat Goldshmidt *1, Iftach Nachman 1
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.

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Biology

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)
Yoray Sharon 1, Lina Alon 1, Sarah Glanz 1, Charlotte Servais 1, Neta Erez 1
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.

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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-Fainaro 1
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.). 

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Medicine

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment
Hadas Bar-Joseph 1,2, Salomon Marcello Stemmer 2,3, Ilan Tsarfaty 2,4, Ruth Shalgi 1,2, Irit Ben-Aharon 2,3
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.

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Biology

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation
Itamar Cohen 1, Yifat Geffen 1, Guy Ravid 1, Tommer Ravid 1
1Department of Biological Chemistry, The Hebrew University of Jerusalem

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

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Immunology and Infection

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis
Nurit P. Azouz 1,2, Mitsunori Fukuda 3, Marc E. Rothenberg 2, Ronit Sagi-Eisenberg 1
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.

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Medicine

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model
Assaf Dotan 1, Israel Kremer 1,2, Orly Gal-Or 1, Tami Livnat 3, Arie Zigler 4, Dan Bourla 1, Dov Weinberger 1,2,3
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).

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Bioengineering

Describing a Transcription Factor Dependent Regulation of the MicroRNA Transcriptome
Uri Rozovski 1, Inbal Hazan-Halevy 2, George Calin 3, David Harris 4, Ping Li 4, Zhiming Liu 4, Michael J. Keating 4, Zeev Estrov 4
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.

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JoVE Journal

Three-dimensional Particle Tracking Velocimetry for Turbulence Applications: Case of a Jet Flow
Jin-Tae Kim 1, David Kim 1, Alex Liberzon 2, Leonardo P. Chamorro 1,3
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.

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Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells
Nadejda Sigal 1, Anna Pasechnek 1, Anat A. Herskovits 1
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.

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Genetics

Generation of Fluorescent Protein Fusions in Candida Species
Sara Gonia 1, Judith Berman 2, Cheryl A. Gale 1
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.

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Cancer Research

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)
Yaara Porat 1, Moshe Giladi 1, Rosa S. Schneiderman 1, Roni Blat 1, Anna Shteingauz 1, Einav Zeevi 1, Mijal Munster 1, Tali Voloshin 1, Noa Kaynan 1, Orna Tal 1, Eilon D. Kirson 2, Uri Weinberg 3, Yoram Palti 2
1Preclinical Research Department, Novocure Ltd., Haifa, Israel, 2Novocure Ltd., Haifa, Israel, 3Novocure GmbH, Lucerne, Switzerland

Tumor Treating Fields (TTFields) are an effective anti-tumor treatment modality delivered via the continuous, noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application to cell lines using a TTFields in vitro application system allows for the determination of the optimal frequency that leads to the highest reduction in cell counts.

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Behavior

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay
Shani Leviatan Ben-Arye 1, Hai Yu 2, Xi Chen 2, Vered Padler-Karavani 1
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.

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Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli
Ilan Edri *1, Michal Goldenberg *1, Michal Lisnyansky 2, Roi Strulovich 2, Hadas Newman 1,3, Anat Loewenstein 1,3, Daniel Khananshvili 2, Moshe Giladi 2,4, Yoni Haitin 2
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.

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JoVE Journal

Imaging FITC-dextran as a Reporter for Regulated Exocytosis
Ofir Klein 1, Amit Roded 1, Koret Hirschberg 2, Mitsunori Fukuda 3, Stephen J. Galli 4, Ronit Sagi-Eisenberg 1
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.

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Behavior

Applying Incongruent Visual-Tactile Stimuli during Object Transfer with Vibro-Tactile Feedback
Jason Friedman 1, Eitan Raveh 2, Tehilla Weiss 2, Sophi Itkin 2, Dafna Niv 2, Mor Hani 2, Sigal Portnoy 2
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.

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Neuroscience

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System
Topaz Altman *1, Roy Maimon *1, Ariel Ionescu *1, Tal Gradus Pery 1, Eran Perlson 1,2
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.

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Engineering

Automated Delivery of Microfabricated Targets for Intense Laser Irradiation Experiments
Yonatan Gershuni 1,2, Michal Elkind 1,2, Dolev Roitman 1,2, Itamar Cohen 1,2, Aviad Tsabary 1,2, Deep Sarkar 1,2, Ishay Pomerantz 1,2
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.

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Genetics

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity
Sampath Kumar Loganathan 1, Ahmad Malik 1,2, Ellen Langille 1,2, Chen Luxenburg 3, Daniel Schramek 1,2
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.

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JoVE Core

T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing
Lael Werner 1,2, Chen Dor 3,4, Naomi Salamon 1, Meital Nagar 3,4, Dror S. Shouval 1,2
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.

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Cancer Research

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs
Neta Moskovits 1,2, Ella Itzhaki 1,2, Nataly Tarasenko 1,2, Eva Chausky 1,2, Avital Bareket-Samish 3, Aleksandr Kaufman 1,2, Raisa Meerson 1,2, Salomon M. Stemmer 1,2,4
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.

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Endless Worms Most Beautiful: Current Methods For Using Nematodes To Study Evolutionary Developmental Biology
Chee Kiang Ewe 1, Pradeep M. Joshi 2, Joel H. Rothman 2
1Department of Neurobiology, Tel Aviv University, 2Department of Molecular, Cellular, Developmental Biology, University of California Santa Barbara

Endless Worms Most Beautiful: Current Methods For Using Nematodes To Study Evolutionary Developmental Biology

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Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures
Sharone Naor 1, Yael Ziv 1, Yosef Shiloh 1
1The David and Inez Myers Laboratory for Cancer Genetics, Department of Human Molecular Genetics and Biochemistry, Faculty of Health and Medical Sciences, School of Medicine, Tel Aviv University

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.

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