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Cell Biology

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 683, Q&A: 0
In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in ...
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Developmental Biology

Protocol for Notch-ligand Binding Assays Using Dynabeads Authors:  Shogo Sawaguchi, Mitsutaka Ogawa and Tetsuya Okajima, date: 10/20/2017, view: 554, Q&A: 0
This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated Dynabeads. Expression of Eogt in Notch1-expressing cells promoted binding toward DLL4-coated ...
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Microbiology

A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions Authors:  Adeline Becquer, Margarita Torres-Aquino, Christine Le Guernevé, Laurie K Amenc, Carlos Trives-Segura, Siobhan Staunton, Hervé Quiquampoix and Claude Plassard, date: 10/20/2017, view: 403, Q&A: 0
In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new ...
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Crude Preparation of Lipopolysaccharide from Helicobacter pylori for Silver Staining and Western Blot Authors:  Hong Li and Mohammed Benghezal, date: 10/20/2017, view: 367, Q&A: 0
This protocol provides an easy and rapid method to prepare lipopolysaccharide from the gastric pathogen Helicobacter pylori for visualization on acrylamide gels by silver staining and for detecting the presence of Lewis antigens by Western blot. The silver staining is a four-step procedure, involving a 20 min-oxidation step, a 10 ...
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Molecular Biology

Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity Authors:  Morten Egevang Jørgensen, Christoph Crocoll, Barbara Ann Halkier and Hussam Hassan Nour-Eldin, date: 10/20/2017, view: 397, Q&A: 0
Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by ...
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NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells Authors:  Alvaro E. Galvis, Hugh E. Fisher and David Camerini, date: 10/20/2017, view: 574, Q&A: 1
This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from ...
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Neuroscience

Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System Authors:  Jianyang Du and Erin E. Koffman, date: 10/20/2017, view: 370, Q&A: 0
A memory trace, also known as a memory engram, is theorized to be a mechanism for physical memory storage in the brain (Silva et al., 2009; Josselyn, 2010) and memory trace is associated with a specific population of neurons (Liu et al., 2012; Ramirez et al., 2013). Labeling and stimulating those neurons will activate ...
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Touchscreen-based Visual Discrimination and Reversal Tasks for Mice to Test Cognitive Flexibility Authors:  Karly M. Turner, Christopher G. Simpson and Thomas H. J. Burne, date: 10/20/2017, view: 309, Q&A: 0
Reversal learning can be used to examine deficits in cognitive flexibility, which have been linked to a number of neuropsychiatric disorders including schizophrenia and addiction. However, methods of examining reversal learning have varied substantially between species. Touchscreen technology has allowed researchers to explore cognitive deficits ...
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Isolation and Purification of Schwann Cells from Spinal Nerves of Neonatal Rat Authors:  Jinkun Wen, Dandan Tan, Lixia Li and Jiasong Guo, date: 10/20/2017, view: 443, Q&A: 0
Primary cultured Schwann cells (SCs) are widely used in the investigation of the biology of SC and are important seed cells for neural tissue engineering. Here, we describe a novel protocol for harvesting primary cultured SCs from neonatal Sprague-Dawley (SD) rats. In the present protocol, dissociated SCs are isolated from the spinal nerves of ...
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Plant Science

Capturing Z-stacked Confocal Images of Living Bacteria Entering Hydathode Pores of Cauliflower Authors:  Aude Cerutti and Alain Jauneau, date: 10/20/2017, view: 469, Q&A: 0
The present protocol to visualize living bacteria at the pore level of cauliflower hydathodes is simple and trained users in confocal microscopy can execute it successfully. It can be easily adapted to capture images with other plant-microorganism interactions at the leaf surface and should be useful to obtain important information on pore and ...
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Histochemical Preparations to Depict the Structure of Cauliflower Leaf Hydathodes Authors:  Aude Cerutti, Marie-Christine Auriac, Laurent D. Noël and Alain Jauneau, date: 10/20/2017, view: 374, Q&A: 0
Hydathodes are plant organs present on leaf margins of a wide range of vascular plants and are the sites of guttation. Both anatomy and physiology of hydathodes are poorly documented. We have recently reported on the anatomy of cauliflower and Arabidopsis thaliana hydathodes and on their infection by the vascular pathogenic bacterium ...
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Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism Authors:  Adeline Becquer, Margarita Torres-Aquino, Christine Le Guernevé, Laurie K Amenc, Carlos Trives-Segura, Siobhan Staunton, Hervé Quiquampoix and Claude Plassard, date: 10/20/2017, view: 366, Q&A: 0
In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the ‘Hartig net’, where exchanges between the two partners take place. ...
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Trimolecular Fluorescence Complementation (TriFC) Assay for Direct Visualization of RNA-Protein Interaction in planta Authors:  Jun Sung Seo and Nam-Hai Chua, date: 10/20/2017, view: 386, Q&A: 0
RNA-Protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA/protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-Protein interactions in living plants have not yet been developed until now. Recently, we have ...
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Monitoring Xylem Hydraulic Pressure in Woody Plants Authors:  Guillaume Charrier, Régis Burlett, Gregory Gambetta, Sylvain Delzon, Jean-Christophe Domec and François Beaujard, date: 10/20/2017, view: 367, Q&A: 0
Xylem sap circulates under either positive or negative hydraulic pressure in plants. Negative hydraulic pressure (i.e., tension) is the most common situation when transpiration is high, and several devices have been developed to quantify it accurately (e.g., Scholander pressure chamber, psychrometers). However, a proper ...
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Stem Cell

In vitro Co-culture of Mesenchymal Stem Cells and Endothelial Colony Forming Cells Authors:  Abbas Shafiee and Kiarash Khosrotehrani, date: 10/20/2017, view: 521, Q&A: 0
The discovery of endothelial colony forming cells (ECFCs) with robust self-renewal and de novo vessel formation potentials suggests that ECFCs can be an excellent cell source for cardiovascular diseases treatment through improving neovascularization in the ischemic tissues. However, their engraftment after transplantation resulted to be low. ...
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