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University of Manitoba

18 ARTICLES PUBLISHED IN JoVE

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Biology

A Gradient-generating Microfluidic Device for Cell Biology
Bong Geun Chung 1, Amir Manbachi 1, Wajeeh Saadi 1, Francis Lin 1, Noo Li Jeon 1, Ali Khademhosseini 1
1Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

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Biology

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands
Rushin Shojaii 1, Stephanie Bacopulos 2,3, Wenyi Yang 2,3, Tigran Karavardanyan 4, Demetri Spyropoulos 5, Afshin Raouf 6, Anne Martel 1,4, Arun Seth 2,3
1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

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Medicine

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Jennifer A. Juno 1, Genevieve Boily-Larouche 1, Julie Lajoie 1, Keith R. Fowke 1,2
1Department of Medical Microbiology, University of Manitoba, 2Department of Community Health Sciences, University of Manitoba

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

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Biology

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System
Emanuel G. Worst 1, Matthias P. Exner 2, Alessandro De Simone 2, Marc Schenkelberger 1, Vincent Noireaux 3, Nediljko Budisa 2, Albrecht Ott 1
1Department of Experimental Physics, Saarland University, 2Institute of Chemistry, Technische Universität Berlin, 3School of Physics and Astronomy, University of Minnesota

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

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Neuroscience

Low-Density Primary Hippocampal Neuron Culture
Reiko T. Roppongi *1,2, Kevin P. Champagne-Jorgensen *1,2, Tabrez J. Siddiqui 1,2
1Department of Physiology and Pathophysiology, University of Manitoba, 2Kleysen Institute for Advanced Medicine, Health Sciences Centre

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

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Immunology and Infection

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood
Ke Yang *1,2,3, Jiandong Wu *3,4, Ling Zhu 1, Yong Liu 1, Michael Zhang 5, Francis Lin 3,4,6,7
1Institute of Applied Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 2University of Science and Technology of China, 3Department of Physics and Astronomy, University of Manitoba, 4Department of Biosystems Engineering, University of Manitoba, 5Seven Oaks General Hospital, 6Department of Immunology, University of Manitoba, 7Department of Biological Sciences, University of Manitoba

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

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Engineering

All-electronic Nanosecond-resolved Scanning Tunneling Microscopy: Facilitating the Investigation of Single Dopant Charge Dynamics
Mohammad Rashidi 1,2, Wyatt Vine 1, Jacob A.J. Burgess 3,4,5, Marco Taucer 1,2,6, Roshan Achal 1, Jason L. Pitters 2, Sebastian Loth 3,4, Robert A. Wolkow 1,2
1Department of Physics, University of Alberta, 2National Institute for Nanotechnology, National Research Council of Canada, Edmonton, 3Max Planck Institute for the Structure and Dynamics of Matter, 4Max Planck Institute for Solid State Research, 5Department of Physics and Astronomy, University of Manitoba, 6Joint Attosecond Science Laboratory, University of Ottawa

We demonstrate an all-electronic method to observe nanosecond-resolved charge dynamics of dopant atoms in silicon with a scanning tunneling microscope.

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Bioengineering

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
Tobias Baumann 1, Franz-Josef Schmitt 2, Almut Pelzer 1, Vivian Jeanette Spiering 3, Georg Johannes Freiherr von Sass 1, Thomas Friedrich 2, Nediljko Budisa 1
1Institute of Chemistry L 1, Department of Biocatalysis, Technical University of Berlin, 2Institute of Chemistry PC 14, Department of Bioenergetics, Technical University of Berlin, 3Institute of Chemistry TC 7, Department of Physical Chemistry/Molecular Material Sciences, Technical University of Berlin

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed "gold" fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

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Bioengineering

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
Jessica H. Nickling *1, Tobias Baumann *1, Franz-Josef Schmitt 2, Maike Bartholomae 3, Oscar P. Kuipers 3, Thomas Friedrich 2, Nediljko Budisa 1
1Department of Biocatalysis, Institute of Chemistry, Technische Universität Berlin, 2Department of Bioenergetics, Institute of Chemistry, Technische Universität Berlin, 3Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Department of Molecular Genetics, University of Groningen

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

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JoVE Journal

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
Javad Alizadeh 1,2, Shahla Shojaei 1,2,3, Simone da Silva Rosa 1, Adel Rezaei Moghadam 1,2, Amir A. Zeki 4, Mohammad Hashemi 5, Marek J. Los 6,7,8, Joseph W. Gordon 1,9, Saeid Ghavami 1,2,10
1Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, 2Biology of Breathing Theme, Children Hospital Research Institute of Manitoba, University of Manitoba, 3Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, 4Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Center for Comparative Respiratory Biology and Medicine, 5Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, 6Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, 7Centre de biophysique moléculaire - UPR 4301, Centre national de la recherche scientifique (CNRS) CS80054, 8LinkoCare Life Sciences AB, 9College of Nursing and Children's Hospital Research Institute of Manitoba, Rady Faculty of Health Sciences, University of Manitoba, 10Health Policy Research Center, Institute of Health, Shiraz University of Medical Sciences

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

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JoVE Core

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum
Shahin Shabanipour 1,2, Azadeh Dalvand 1,2, Xiaodan Jiao 1,2, Maryam Rahimi Balaei 1,2, Seung H. Chung 3, Jiming Kong 1, Marc. R. Del Bigio 2,4, Hassan Marzban 1,2
1Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 2The Children's Hospital Research Institute of Manitoba (CHRIM), Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 3Department of Oral Biology, University of Illinois at Chicago, 4Department of Pathology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

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Chemistry

A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes
Franklin Brian Apea-Bah 1,2, Pamela Drawbridge 1,2, Trust Beta 1,2
1Department of Food and Human Nutritional Sciences, University of Manitoba, 2Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba

Phenolic acids are important phytochemicals that are present in whole grains. They possess bioactive properties such as antioxidant protective functions. This work aimed at reporting on a generalized method for the HPLC identification, total phenolic content estimation, and determination of the antioxidant capacity of phenolic acids in cereals and legumes.

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Neuroscience

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo
Jessica Meza-Resillas *1, Noushin Ahmadpour *1, Michael Stobart 1, Jillian Stobart 1
1College of Pharmacy, Rady Faculty of Health Sciences, University of Manitoba

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

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Immunology and Infection

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices
Ali N. Doucet 1, Carmine J. Slipski 1, George R. Golding 1,2, Michael R. Mulvey 1,2, Denice C. Bay 1
1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, 2National Microbiology Laboratory, Public Health Agency of Canada

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

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Bioengineering

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability
Tuyet Mai Thi To 1, Vladimir Kubyshkin 2, Franz-Josef Schmitt 3, Nediljko Budisa 1,2, Thomas Friedrich 1
1Institute of Chemistry, Technische Universität Berlin, 2Department of Chemistry, University of Manitoba, 3Institute of Physics, Martin-Luther-Universität Halle-Wittenberg

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

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Neuroscience

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons
Noriko Koganezawa 1, Reiko T. Roppongi 2, Yuko Sekino 3,4, Izuo Tsutsui 3, Ayaka Higa 5, Tomoaki Shirao 1,5
1Department of Pharmacology, Graduate School of Medicine, Gunma University, 2Gunma University Initiative for Advanced Research, Gunma University, 3Department of Veterinary Pathophysiology and Animal Health, Graduate school of Agricultural and Life Sciences, The University of Tokyo, 4Institute for Drug Discovery Innovation, 5AlzMed, Inc.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

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Environment

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration
Kadir Yanaç 1, Jhannelle Francis 2, Jocelyn Zambrano-Alvarado 2, Qiuyan Yuan 1, Miguel Uyaguari-Díaz 2
1Department of Civil Engineering, Price Faculty of Engineering, University of Manitoba, 2Department of Microbiology, Faculty of Science, University of Manitoba

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

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Immunology and Infection

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System
Dawn White 1, Ayush Kumar 1
1Department of Microbiology, University of Manitoba

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

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