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Past Issues in 2017

Volume: 7 Issue: 24

Biochemistry

A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles Authors:  Rubul Mout and Vincent M. Rotello, date: 12/20/2017, view: 5899, Q&A: 0
In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and ...
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Cancer Biology

FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand Authors:  Jessica Tome-Garcia, Fiona Doetsch and Nadejda M. Tsankova, date: 12/20/2017, view: 9825, Q&A: 0
Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma ...
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Cell Biology

Using xCELLigence RTCA Instrument to Measure Cell Adhesion Authors:  Hellyeh Hamidi, Johanna Lilja and Johanna Ivaska, date: 12/20/2017, view: 9744, Q&A: 0
Cell adhesion to neighbouring cells and to the underlying extracellular matrix (ECM) is a fundamental requirement for the existence of multicellular organisms. As such, the formation, stability and dissociation of cell adhesions are subject to tight control in space and time and perturbations within the sophisticated adhesion machinery are ...
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Immunogold Electron Microscopy of the Autophagosome Marker LC3 Authors:  Domenico Mattoscio, Andrea Raimondi, Carlo Tacchetti and Susanna Chiocca, date: 12/20/2017, view: 9902, Q&A: 0
Even though autophagy was firstly observed by transmission electron microscopy already in the 1950s (reviewed in Eskelinen et al., 2011), nowadays this technique remains one of the most powerful systems to monitor autophagic processes. The autophagosome, an LC3-positive double membrane structures enclosing cellular materials, represents ...
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Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue Authors:  Alicia A Cutler, Anita H Corbett and Grace K Pavlath, date: 12/20/2017, view: 8920, Q&A: 0
Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. ...
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Developmental Biology

Ex vivo Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery Authors:  Damayanti Chakraborty, Masanaga Muto and Michael J. Soares, date: 12/20/2017, view: 5566, Q&A: 0
In this protocol report, we describe a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Lentiviral packaged gene constructs can be efficiently and specifically delivered to the trophoblast cell lineage of the blastocyst. The consequences of ‘gain-of-function’ and ‘loss-of-function’ blastocyst ...
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Immunology

Analysing Temporal Dynamics of T Cell Division in vivo Using Ki67 and BrdU Co-labelling by Flow Cytometry Authors:  Thea Hogan, Andrew Yates and Benedict Seddon, date: 12/20/2017, view: 9045, Q&A: 0
This protocol was developed to increase the richness of information available from in vivo T cell proliferation studies. DNA labelling techniques such as BrdU incorporation allow precise control of label administration and withdrawal, so that the division history of a population can be tracked in detail over long timeframes (days-weeks). ...
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Generation of Busulfan Chimeric Mice for the Analysis of T Cell Population Dynamics Authors:  Thea Hogan, Andrew Yates and Benedict Seddon, date: 12/20/2017, view: 7784, Q&A: 0
This protocol was developed to generate chimeric mice in which T lymphocytes could be stratified by age on the basis of congenic marker expression. The conditioning drug busulfan is used to ablate host haematopoietic stem cells while leaving the peripheral immune system intact. Busulfan treatment is followed by bone marrow transplantation (BMT), ...
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Mouse Model of Immune Complex-mediated Vasculitis in Dorsal Skin and Assessment of the Neutrophil-mediated Tissue Damage Authors:  Dongsheng Jiang, Juliane C. de Vries, Jana Muschhammer, Anca Sindrilaru and Karin Scharffetter-Kochanek, date: 12/20/2017, view: 5731, Q&A: 0
Neutrophils are the most abundant leukocytes in the blood. In the recent decades, their crucial roles in host defense, immune regulation and tissue damage have been studied in a deeper dimension. In this protocol, we described a mouse model of immune complex-mediated vasculitis in the dorsal skin induced by Arthus reaction, and the subsequent ...
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Microbiology

Rice Black-streaked Dwarf Virus Preparation and Infection on Rice Authors:  Zongtao Sun, Hehong Zhang, Kaili Xie, Xiaoxiang Tan, Hengmu Zhang and Jianping Chen, date: 12/20/2017, view: 6785, Q&A: 1
Rice black-streaked dwarf virus (RBSDV), a member of genus Fijivirus in the family Reoviridae, infects rice, maize, barley and wheat, and can seriously affect crop yields. RBSDV is transmitted by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner. RBSDV has 10 linear dsRNA genomic segments, ...
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Oral Microbiome Characterization in Murine Models Authors:  Loreto Abusleme, Bo-Young Hong, Anilei Hoare, Joanne E. Konkel, Patricia I. Diaz and Niki M. Moutsopoulos, date: 12/20/2017, view: 6794, Q&A: 0
The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe ...
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Molecular Biology

Genotyping-free Selection of Double Allelic Gene Edited Medaka Using Two Different Fluorescent Proteins Authors:  Yu Murakami, Satoshi Ansai, Akari Yonemura and Masato Kinoshita, date: 12/20/2017, view: 5272, Q&A: 0
This protocol describes a simple genotyping using two different colors of fluorescent protein genes inserted at the target locus. This method makes it possible to determine the genotype of each individual simply by observing the fluorescence later than F1 generation.
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Neuroscience

Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes Authors:  Jigar Modi, Howard Prentice and Jang-Yen Wu, date: 12/20/2017, view: 4339, Q&A: 0
In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational ...
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Plant Science

Preparation of Onion Epidermal Cell Walls for Imaging by Atomic Force Microscopy (AFM) Authors:  Tian Zhang and Daniel J. Cosgrove, date: 12/20/2017, view: 11377, Q&A: 0
The growing plant cell wall is comprised of long, thin cellulose microfibrils embedded in a hydrated matrix of polysaccharides and glycoproteins. These components are typically constructed in layers (lamellae) on the inner surface of the cell wall, i.e., between the existing wall and the plasma membrane. The organization of these ...
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Determination of H+-ATPase Activity in Arabidopsis Guard Cell Protoplasts through H+-pumping Measurement and H+-ATPase Quantification Authors:  Shota Yamauchi and Ken-ichiro Shimazaki, date: 12/20/2017, view: 6739, Q&A: 0
The opening of stomata in plants in response to blue light is driven by the plasma membrane H+-ATPase in guard cells. To evaluate the activation of the H+-ATPase in vivo, we can use H+-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of ...
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Assessing Rates of Long-distance Carbon Transport in Arabidopsis by Collecting Phloem Exudations into EDTA Solutions after Photosynthetic Labeling with [14C]CO2 Authors:  Umesh P Yadav, Aswad S Khadilkar, Mearaj A Shaikh, Robert Turgeon and Brian G Ayre, date: 12/20/2017, view: 4679, Q&A: 0
Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of ...
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Assessing Long-distance Transport from Photosynthetic Source Leaves to Heterotrophic Sink Organs with [14C]CO2 Authors:  Umesh P Yadav, Aswad S Khadilkar, Mearaj A Shaikh, Robert Turgeon and Brian G Ayre, date: 12/20/2017, view: 4451, Q&A: 0
Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of ...
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Quantifying the Capacity of Phloem Loading in Leaf Disks with [14C]Sucrose Authors:  Umesh P Yadav, Aswad S Khadilkar, Mearaj A Shaikh, Robert Turgeon and Brian G Ayre, date: 12/20/2017, view: 5225, Q&A: 0
Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of ...
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Biomechanical Characterization of Onion Epidermal Cell Walls Authors:  Daniel M. Durachko, Yong Bum Park, Tian Zhang and Daniel J. Cosgrove, date: 12/20/2017, view: 9426, Q&A: 1
Here we describe two experimental protocols to measure the biomechanical properties of primary (growing) plant cell walls, with a focus on analyzing cell wall epidermal strips of onion scales. The first protocol measures cell wall creep (time-dependent irreversible extension) under constant force. Such creep is often mediated by the wall-loosening ...
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Infection of Soybean Plants with the Insect Bacterial Symbiont Burkholderia gladioli and Evaluation of Plant Fitness Authors:  Paul Gaube, Martin Kaltenpoth and Laura V. Flórez, date: 12/20/2017, view: 5243, Q&A: 0
To investigate the establishment and consequences of host-microbe interactions, it is important to develop controlled infection assays suitable for each system, as well as appropriate methods to evaluate successful infection and its associated effects. Here, we describe a procedure for bacterial inoculation of soybean plants, followed by the ...
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