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The Hebrew University of Jerusalem

34 ARTICLES PUBLISHED IN JoVE

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Biology

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation
Oshri Avraham *1, Sophie Zisman *1, Yoav Hadas 1, Lilach Vald 1, Avihu Klar 1
1Department of Medical Neurobiology, Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

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Biology

Photobleaching Assays (FRAP & FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells
Malka Nissim-Rafinia 1, Eran Meshorer 1
1Department of Genetics, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

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Chemistry

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations
Ido Braslavsky 1,2, Ran Drori 1
1Institute of Biochemistry, Food Science, and Nutrition , The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, 2Department of Physics and Astronomy, Ohio University

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

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Biology

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
Oswald J. Schmitz 1, Mark A. Bradford 1, Michael S. Strickland 1,2, Dror Hawlena 3
1School of Forestry and Environmental Studies, Yale University, 2Department of Biological Sciences, Virginia Tech, 3Department of Ecology, Evolution and Behavior, The Hebrew University of Jerusalem

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

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Neuroscience

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo
Ayelet Kohl 1, Yoav Hadas 2, Avihu Klar 2, Dalit Sela-Donenfeld 1
1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, 2Department of Medical Neurobiology, The Hebrew University of Jerusalem

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox–plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

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Behavior

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome
Shahar Barbash 1, Geula Hanin 1, Hermona Soreq 1
1Department of Biological Chemistry and The Edmond & Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

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Chemistry

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Michal S. Shoshan 1, Edit Y. Tshuva 1, Deborah E. Shalev 2
1Department of Chemistry, The Hebrew University of Jerusalem, 2Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

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Chemistry

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay
Nitzan Ganot *1, Sigalit Meker *1, Lilia Reytman *1, Avia Tzubery *1, Edit Y. Tshuva 1
1Institute of Chemistry, The Hebrew University of Jerusalem

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

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Engineering

Monolayer Contact Doping of Silicon Surfaces and Nanowires Using Organophosphorus Compounds
Ori Hazut 1,2, Arunava Agarwala 1,2, Thangavel Subramani 1,2, Sharon Waichman 1,2, Roie Yerushalmi 1,2
1Institute of Chemistry, The Hebrew University of Jerusalem, 2Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem

A detailed procedure for surface doping of Silicon interfaces is provided. The ultra-shallow surface doping is demonstrated by using phosphorus containing monolayers and rapid annealing process. The method can be used for doping of macroscopic area surfaces as well as nanostructures.

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Education

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides
Sivan Yuran 1, Meital Reches 1
1Institute of Chemistry and The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

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Chemistry

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity
Koli Basu 1, Christopher P. Garnham 2, Yoshiyuki Nishimiya 3, Sakae Tsuda 3, Ido Braslavsky 4, Peter Davies 1
1Department of Biomedical and Molecular Sciences, Queen's University, 2National Institute of Neurological Disorders and Stroke, Porter Neuroscience Research Center, 3Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology, 4The Robert H. Smith Faculty of Agriculture, Food and Environment, Institute of Biochemistry, Food Science, and Nutrition, The Hebrew University of Jerusalem

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

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Neuroscience

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye
Amy E. Birsner *1, Ofra Benny *2, Robert J. D'Amato 1,3
1Vascular Biology Program, Boston Children's Hospital, 2Institute for Drug Research, School of Pharmacy, The Hebrew University of Jerusalem, 3Department of Ophthalmology, Harvard Medical School

The protocol describes the corneal micropocket assay as developed in mice.

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Immunology and Infection

ScanLag: High-throughput Quantification of Colony Growth and Lag Time
Irit Levin-Reisman 1, Ofer Fridman 1, Nathalie Q. Balaban 1
1Racah Institute of Physics, The Hebrew University of Jerusalem

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

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Education

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow
Uri Green 1,2, Yulia Shenberger 3, Zeev Aizenshtat 1, Haim Cohen 2,4, Sharon Ruthstein 3
1Chemistry Institute, The Hebrew University of Jerusalem, 2Biological Chemistry Department, Ariel University, 3Chemistry Department, Faculty of Exact Science, Bar Ilan University, 4Chemistry Department, Ben-Gurion University

Stable radicals that are present in carbon substrates interact with paramagnetic oxygen through a Heisenberg spin exchange. This interaction can be significantly reduced under STP conditions by flowing a diamagnetic gas over the carbon system. This manuscript describes a simple method to characterize the nature of those radicals.

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Behavior

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience
Hagit Turm 1, Diptendu Mukherjee 1, Doron Haritan 1, Maayan Tahor 1, Ami Citri 1
1The Alexander Silberman Institute of Life Sciences & Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

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Biology

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example
Arava Shatil-Cohen 1, Hadas Sibony 1, Xavier Draye 2, François Chaumont 3, Nava Moran 1, Menachem Moshelion 1
1The RH Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, 2Earth and Life Institute, Université catholique de Louvain, 3Institut des Sciences de la Vie, Université catholique de Louvain

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

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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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Biology

Identifying Protein-protein Interaction Sites Using Peptide Arrays
Hadar Amartely 1, Anat Iosub-Amir 1, Assaf Friedler 1
1Institute of Chemistry, The Hebrew University of Jerusalem

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

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Chemistry

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy
Priyadip Das 1, Tal Duanias-Assaf 1, Meital Reches 1
1Institute of Chemistry and The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

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JoVE Journal

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry
Rosi Fassler *1, Nufar Edinger *1, Oded Rimon 1, Dana Reichmann 1
1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram, The Hebrew University of Jerusalem

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

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Biochemistry

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization
Hadar Amartely 1, Daniel Some 2, Ayala Tsadok 3, Mario Lebendiker 1
1Wolfson Centre for Applied Structural Biology, The Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem, 2Wyatt Technology Corporation, 3Danyel Biotech Ltd

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

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Biochemistry

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)
Daniel Some 1, Hadar Amartely 2, Ayala Tsadok 3, Mario Lebendiker 2
1Wyatt Technology Corporation, 2Wolfson Centre for Applied Structural Biology, The Alexander Silberman Institute of Life Science, The Hebrew University of Jerusalem, 3Danyel Biotech Ltd.

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

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Chemistry

Calcium Carbonate Formation in the Presence of Biopolymeric Additives
David N. Azulay 1,2, Liraz Chai 1,2
1Institute of Chemistry, Edmond J. Safra Campus, The Hebrew University of Jerusalem, 2The Harvey M. Krueger Family Center for Nanoscience and Nanotechnology, Edmond J. Safra Campus, The Hebrew University of Jerusalem

We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

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Behavior

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring
Liat Morgan 1, Tal Raz 1
1Koret School of Veterinary Medicine, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

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Genetics

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model
Keerat Kaur 1,2,3, Nishat Sultana 1,2,3, Yoav Hadas 1,2,3, Ajit Magadum 1,2,3, Mohammad Tofael Kabir Sharkar 1,2,3, Elena Chepurko 1,2,3, Lior Zangi 1,2,3
1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, 2Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 3Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai

This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.

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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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Environment

A Telemetric, Gravimetric Platform for Real-Time Physiological Phenotyping of Plant–Environment Interactions
Ahan Dalal 1, Itamar Shenhar 1, Ronny Bourstein 1, Amir Mayo 1, Yael Grunwald 1, Nir Averbuch 1, Ziv Attia 1, Rony Wallach 1, Menachem Moshelion 1
1Faculty of Agriculture, Food and Environment, The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem

This high-throughput, telemetric, whole-plant water relations gravimetric phenotyping method enables direct and simultaneous real-time measurements, as well as the analysis of multiple yield-related physiological traits involved in dynamic plant–environment interactions.

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Biochemistry

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One α-Synuclein Monomer at a Time
Sofia Zaer 1, Eitan Lerner 1,2
1Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus, The Hebrew University of Jerusalem, 2The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in α-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

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Neuroscience

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains
Ron Refaeli 1, Inbal Goshen 1
1Edmond and Lily Safra Center for Brain Sciences (ELSC), The Hebrew University of Jerusalem

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

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Chemistry

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization
Ran Drori 1,2, Yitzhar Shalom 1,2
1Department of Chemistry and Biochemistry, Yeshiva University, 2Department of Physics, Katz School of Science and Health, Yeshiva University

The present protocol describes the crystallization of microscopic ice crystals and clathrate hydrates in microfluidic devices, enabling liquid exchange around the formed crystals. This provides unparalleled possibilities to examine the crystallization process and binding mechanisms of the inhibitors.

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Cancer Research

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography
Dimitri Gaber 1, Michal Aharoni-Simon 1, Ortal Zaks 1, Keren Ben-Yaakov 1, Ziv Rotfogel 1,2, Hana Leiba 1,2, Avital Eisenberg-Lerner *1, Arie L. Marcovich *1,2
1Ophthalmology Research Laboratory, Kaplan Medical Center, 2Department of Ophthalmology, Kaplan Medical Center, Faculty of Medicine, The Hebrew University of Jerusalem

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

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Cancer Research

Pancreatic Tissue Dissection to Isolate Viable Single Cells
Oshri Yosefov-Levi 1, Sharona Tornovsky 1, Oren Parnas 1
1The Lautenberg Center for Immunology and Cancer Research, The Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University of Jerusalem

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

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Biology

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes
Vijigisha Srivastava 1, Einav Gross 1
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, Institute for Medical Research, Israel-Canada (IMRIC), The Hebrew University of Jerusalem

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

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Biology

Imaging Flow Cytometry to Study Microbial Autoaggregation
Ronit Suissa 1, Uzi Hadad 2, Michael Meijler 1, Ilana Kolodkin-Gal 3,4
1Department of Chemistry, Ben-Gurion University of the Negev, 2Ilse Kats Institute for Nanoscale Science & Technology, Ben-Gurion University of the Negev, 3Department of Plant Pathology and Microbiology, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, 4The Scojen institute for synthetic biology, Reichman university

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

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