Volume: 5 Issue: 12 —BIO-PROTOCOL

Cancer Biology

Metabolic Assays for Detection of Neutral Fat Stores Authors: Jan M. Baumann, Leila Kokabee, Xianhiu Wang, Yan Sun, Jason Wong and Douglas S. Conklin, date: 06/20/2015, view: 14068, Q&A: 1

Lipid droplets (LDs) are ubiquitous intracellular structures whose formation, growth, and maintenance are highly regulated (Wang et al., 2013; Ranall et al., 2011; Goodman, 2009). Lipid metabolism and droplet dynamics are of considerable interest to agriculture, biofuel production, viral pathology, nutrition, and cancer biology ...

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Immunology

Ex vivo Human Natural Killer (NK) Cell Stimulation and Intracellular IFNγ and CD107a Cytokine Staining Authors: Vanessa A. York and Jeffrey M. Milush, date: 06/20/2015, view: 16382, Q&A: 1

Natural killer (NK) cells comprise 5–20% of peripheral blood mononuclear cells (PBMC) in humans. In addition to their fundamental roles in the defense against viral infections and tumor surveillance, NK cells help shape adaptive immune responses through their production of cytokines. NK cells are traditionally identified as CD3^neg, CD14 ...

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Non-invasive Intratracheal Instillation in Mice Authors: Guadalupe Ortiz-Muñoz and Mark R. Looney, date: 06/20/2015, view: 17049, Q&A: 1

The intratracheal instillation technique is used to deliver a variety of agents to the lungs ranging from pathogens (bacteria, viruses), toxins, to therapeutic agents. To model lung inflammation and injury, LPS can be administrated via intranasal, intratracheal, or aerosol approaches. Each technique has its limitations. The intratracheal technique ...

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Two-event Transfusion-related Acute Lung Injury Mouse Model Authors: Guadalupe Ortiz-Muñoz and Mark R. Looney, date: 06/20/2015, view: 8226, Q&A: 0

Transfusion-related acute lung injury (TRALI) is defined as acute lung injury that occurs within 6 hours of a blood product transfusion. TRALI continues to be a leading cause of transfusion-related mortality and we have developed a mouse model of TRALI to better understand the mechanisms by which injury occurs and to test therapeutic approaches. ...

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Microbiology

Generating Isogenic Deletions (Knockouts) in Francisella tularensis, a Highly-infectious and Fastidious Gram-negative Bacterium Authors: Xiaojun Wu, Guoping Ren and Jason F. Huntley, date: 06/20/2015, view: 9164, Q&A: 0

Generating bacterial gene deletion mutants, also known as knockouts (KOs), is a powerful tool to investigate individual gene functions. However, fastidious bacteria such as Francisella tularensis (F. tularensis) often are difficult to genetically manipulate. Indeed, many different approaches have been tested to generate F. ...

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Visual Assessment of the Severity of Fusarium Seedling Blight (FSB) and Fusarium Head Blight (FHB) Disease in Barley Authors: Shahin S. Ali, Lokanadha R. Gunupuru and Fiona M. Doohan, date: 06/20/2015, view: 9143, Q&A: 0

Fusarium pathogens are among the most damaging pathogens of cereals. These pathogens have the ability to attack the roots, seedlings, and flowering heads of barley and wheat plants (Simpson et al., 2004). Resulting in yield loss and head blight disease and also resulting in the contamination of grain with mycotoxins harmful to ...

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Mismatched Primer Extension Assays Authors: Vasudevan Achuthan and Jeffrey J. DeStefano, date: 06/20/2015, view: 6906, Q&A: 0

Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3ʹ end is a major strength of the mismatched primer extension assays. Recently, we used ...

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Primer Extension Reactions for the PCR- based α- complementation Assay Authors: Vasudevan Achuthan and Jeffrey J. DeStefano, date: 06/20/2015, view: 9837, Q&A: 0

The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus ...

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Neuroscience

Electroporation of Embryonic Chick Eyes Authors: Agustín Luz-Madrigal, Erika Grajales-Esquivel and Katia Del Rio-Tsonis, date: 06/20/2015, view: 12256, Q&A: 0

The chick embryo has prevailed as one of the major models to study developmental biology, cell biology and regeneration. From all the anatomical features of the chick embryo, the eye is one of the most studied. In the chick embryo, the eye develops between 26 and 33 h after incubation (Stages 8-9, Hamburger and Hamilton, 1951). It originates from ...

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Plant Science

Gentiobiose Feeding in Gentian in vitro Overwintering Buds or Plantlets Authors: Hideyuki Takahashi and Masahiro Nishihara, date: 06/20/2015, view: 7129, Q&A: 0

To study the functions of sugars in plants, feeding experiment is one of the most common and easy methods. However, the traditional method, e.g., a floating of leaf discs on sugar-containing solution seems to have an insufficient efficiency of sugar uptake, despite of high osmotic and injury effects. This is a protocol to feed ...

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A Simple Protocol for the Immunolabelling of Arabidopsis Pollen Tube Membranes and Cell Wall Polymers Authors: Marie Dumont, Céline Cataye, Arnaud Lehner, Eric Maréchal, Patrice Lerouge, Denis Falconet and Jean-Claude Mollet, date: 06/20/2015, view: 10327, Q&A: 0

The pollen tube, a fast tip-growing cell, is an excellent model to study membrane and cell wall biosynthesis. Here, we describe a simple protocol using an easy to use device to perform immunofluorescence labelling of pollen tube membrane and cell wall. The use of the NucleoSpin column to perform all the steps of the immunolabelling procedure ...

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Analysis of Sugar Component of a Hot Water Extract from Arabidopsis thaliana Pollen Tubes Using GC-EI-MS Authors: Marie Dumont, Arnaud Lehner, Corinne Loutelier-Bourhis, Jean-Claude Mollet and Patrice Lerouge, date: 06/20/2015, view: 7573, Q&A: 0

Extraction with hot water is the oldest and simplest method used to recover pectin from an alcohol insoluble residue extract, although this method has not been widely used for the cell wall analysis of pollen tube, a model used to study cell wall. This protocol described this method applied for pectin extraction from 6 h-old Arabidopsis ...

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Virus-induced Gene Silencing (VIGS) in Barley Seedling Leaves Authors: Lokanadha R. Gunupuru, Shahin S. Ali, Fiona M. Doohan and Steven R. Scofield, date: 06/20/2015, view: 11947, Q&A: 1

Virus induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterisation. This method exploits a dsRNA-mediated antiviral defence mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid time frame as compared to the generation of ...

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Development and Implementation of an in vitro Culture System for Intact Detached Grape Berries Authors: Zhanwu Dai, Messa Meddar, Serge Delrot and Eric Gomès, date: 06/20/2015, view: 10537, Q&A: 0

Grape composition depends on the metabolites accumulated and synthesized during grape development. It is of paramount importance for grape growers because of its major role in shaping wine quality. Therefore, understanding the regulation mechanisms that control the accumulation of quality-related metabolites in grape is of both scientific and ...

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Past Issues in 2015

Volume: 5 Issue: 12

Cancer Biology

Immunology

Microbiology

Neuroscience

Plant Science