Volume: 8 Issue: 16 —BIO-PROTOCOL

Biochemistry

Modifying Styrene-maleic Acid Co-polymer for Studying Lipid Nanodiscs by Direct Fluorescent Labeling Authors: Victoria Schmidt and James N. Sturgis, date: 08/20/2018, view: 4342, Q&A: 0

This protocol was developed to functionalize styrene maleic acid (SMA) by direct fluorescent labeling in an easy way, accessible to biochemistry laboratories. This novel method is based on the coupling of carboxylic acids to primary amines using a carbodiimide, a reaction commonly used for protein chemistry. The procedure uses the hydrolyzed ...

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Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells Authors: Archana Varadaraj, Carina Magdaleno and Karthikeyan Mythreye, date: 08/20/2018, view: 3857, Q&A: 0

Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell ...

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Immunology

Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry Authors: Katrina Kildey, Becker M.P. Law, Kimberly A. Muczynski, Ray Wilkinson, Helen Healy and Andrew J. Kassianos, date: 08/20/2018, view: 6019, Q&A: 0

Inflammatory immune cells play direct pathological roles in cases of acute kidney injury (AKI) and chronic kidney disease (CKD). However, the identification and characterization of distinct populations of leukocytes in human kidney biopsies have been confounded by the limitations of immunohistochemical (IHC)-based techniques used to detect them. ...

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Microbiology

Microfluidics-Based Analysis of Contact-dependent Bacterial Interactions Authors: Robert Cooper, Lev Tsimring and Jeff Hasty, date: 08/20/2018, view: 4514, Q&A: 0

Bacteria in nature live in complex communities with multiple cell types and spatially-dependent interactions. Studying cells in well-mixed environments such as shaking culture tubes or flasks cannot capture these spatial dynamics, but cells growing in full-fledged biofilms are difficult to observe in real time. We present here a protocol for ...

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Assessment of Caenorhabditis elegans Competitive Fitness in the Presence of a Bacterial Parasite Authors: McKenna. J. Penley and Levi T. Morran, date: 08/20/2018, view: 3859, Q&A: 0

Accurate measurements of an organism’s fitness are crucial for measuring evolutionary change. Methods of fitness measurement are most accurate when incorporating an individual’s survival and fecundity, as well as accounting for any ecological interactions or environmental effects experienced by the organism. Here, we describe a protocol for ...

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In vivo and in vitro ³¹P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals Authors: Christine Le Guernevé, Adeline Becquer, Margarita Torres-Aquino, Laurie K Amenc, Carlos Trives-Segura, Siobhan Staunton, Claude Plassard and Hervé Quiquampoix, date: 08/20/2018, view: 3777, Q&A: 0

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (³¹P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or ...

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Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor Authors: Helen Wang, Samuel S. Slattery, Bogumil J. Karas and David R. Edgell, date: 08/20/2018, view: 5173, Q&A: 2

Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of ...

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Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone Authors: Jeffrey W. Perry and Andrew W. Tai, date: 08/20/2018, view: 3596, Q&A: 0

Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to ...

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CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus Authors: Manuel V. Borca, Keith A. Berggren, Elizabeth Ramirez-Medina, Elizabeth A. Vuono and Douglas P. Gladue, date: 08/20/2018, view: 5899, Q&A: 0

Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of ...

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Liposome Flotation Assay for Studying Interactions Between Rubella Virus Particles and Lipid Membranes Authors: Kyoko Saito, Noriyuki Otsuki, Makoto Takeda and Kentaro Hanada, date: 08/20/2018, view: 6450, Q&A: 0

Rubella virus (RuV) is an enveloped, positive-sense single-stranded RNA virus that is pathogenic to humans. RuV binds to the target cell via the viral envelope protein E1, but the specific receptor molecules on the target cell are yet to be fully elucidated. Here, we describe a protocol for liposome flotation assay to study direct interactions ...

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Neuroscience

Image-Based Analysis of Mitochondrial Area and Counting from Adult Mouse Dopaminergic Neurites Authors: Nadee Nissanka, Carlos T Moraes and Mlena Pinto, date: 08/20/2018, view: 6205, Q&A: 0

Mitochondria form dynamic cytoplasmic networks which undergo morphological changes in order to adapt to cellular stresses and signals. These changes can include alterations in size and number within a given cell. Analysis of the whole network can be a useful metric to assess overall mitochondrial health, particularly in neurons, which are highly ...

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Trace Fear Conditioning: Procedure for Assessing Complex Hippocampal Function in Mice Authors: Vijendra Sharma, Noah Cohen, Rapita Sood, Hadile Ounallah-Saad, Shunit Gal-Ben-Ari and Kobi Rosenblum, date: 08/20/2018, view: 6263, Q&A: 0

The trace fear conditioning protocol is designed to measure hippocampal function in mice. The protocol includes a neutral conditioned stimulus (tone) and an aversive unconditioned stimulus (shock), separated in time by a trace interval. The trace interval between the tone and the shock critically involves the hippocampus and could be used to ...

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Studying the Role of Microglia in Neurodegeneration and Axonal Regeneration in the Murine Visual System Authors: Alexander M Hilla and Dietmar Fischer, date: 08/20/2018, view: 7463, Q&A: 0

Microglia reside in the central nervous system (CNS) and are involved in the maintenance of the physiologic state. They constantly survey their environment for pathologic alterations associated with injury or diseases. For decades, researchers have investigated the role of microglia under different pathologic conditions, using approaches aiming to ...

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Plant Science

An Inexpensive and Comprehensive Method to Examine and Quantify Field Insect Community Influenced by Host Plant Olfactory Cues Authors: Rupesh Kariyat, Jesus Chavana and Jasleen Kaur, date: 08/20/2018, view: 3863, Q&A: 0

Insect pollinators, herbivores and their natural enemies use olfactory cues emitted by their host plants to locate them. In insect-plant ecology, understanding the mechanisms underlying these interactions are of critical importance, as this bio-communication has both ecological and agricultural applications. However, the first step in such ...

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Pollen Germination and Pollen Tube Growth of Arabidopsis thaliana: In vitro and Semi in vivo Methods Authors: Hugh Dickinson, Josefina Rodriguez-Enriquez and Robert Grant-Downton , date: 08/20/2018, view: 11375, Q&A: 2

Studies of pollen germination and post-germination development are not only essential for understanding plant reproduction but also are an excellent model system for tip-based growth. Here we describe easy, reproducible methods for germination and growth of pollen from the model plant Arabidopsis thaliana in artificial conditions. Our ...

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Stem Cell

Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates Authors: Elena Redondo-Castro, Catriona J Cunningham, Jonjo Miller, Stuart A Cain, Stuart M Allan and Emmanuel Pinteaux, date: 08/20/2018, view: 6321, Q&A: 0

The 3D culture of human mesenchymal stem cells (hMSCs) represents a more physiological environment than classical 2D culture and has been used to enhance the MSC secretome or extend cell survival after transplantation. Here we describe a simple and affordable method to generate 3D spheroids of hMSCs by seeding them at high density in a low-binding ...

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Systems Biology

Quantitative ChIP-seq by Adding Spike-in from Another Species Authors: Kongyan Niu, Rui Liu and Nan Liu, date: 08/20/2018, view: 17337, Q&A: 0

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of ...

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Past Issues in 2018

Volume: 8 Issue: 16

Biochemistry

Immunology

Microbiology

Neuroscience

Plant Science

Stem Cell

Systems Biology