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Past Issues in 2016

Volume: 6 Issue: 18

Immunology

Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia Authors: Anupam Jhingran, Shinji Kasahara and Tobias M Hohl, date: 09/20/2016, view: 11411, Q&A: 0

Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding ...

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Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells Authors: Loredana Cifaldi, Franco Locatelli and Doriana Fruci, date: 09/20/2016, view: 4683, Q&A: 0

Natural killer (NK) cells play key roles in innate and adaptive immune responses against virus and tumor cells. Their function relies on the dynamic balance between activating and inhibiting signals through receptors that bind ligands expressed on target cells. The absence of inhibitory receptor engagement with their ligands and the presence of ...

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Peptide Loading on MHC Class I Molecules of Tumor Cells Authors: Loredana Cifaldi, Franco Locatelli and Doriana Fruci, date: 09/20/2016, view: 7469, Q&A: 0

MHC class I molecules present peptides to cytotoxic T cells allowing the immune system to scan for intracellular pathogens and mutated proteins. The generation of antigenic peptides is a multistep process that ends in the endoplasmic reticulum (ER). Only peptides with the right length and sequence will bind nascent MHC class I molecules in the ER. ...

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Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents Authors: Patricia Dillenburg-Pilla, Carlos R. Zárate-Bladés, Phyllis B. Silver, Reiko Horai and Rachel R. Caspi, date: 09/20/2016, view: 6899, Q&A: 0

The contribution of microbiota in regulating multiple physiological and pathological host responses has been studied intensively in recent years. Evidence suggests that commensal microbiota can directly modulate different populations of cells of the immune system (e.g., Ivanov et al., 2008; Atarashi et al., 2011). ...

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Imaging Thick Lymph Node Tissue Sections Authors: Jyh Liang Hor and Scott N. Mueller, date: 09/20/2016, view: 9416, Q&A: 0

Our protocol describes a simple procedure for imaging thick lymph node sections by 2-photon microscopy. Lymph nodes are sectioned using a vibratome (vibrating microtome) to produce slices of tissue that can then be stained with fluorescently labeled antibodies. The thick tissue sections (150-200 μm depth) allow for the detection of cell clustering ...

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Skin TRITC Painting to Track Dendritic Cells Migrating to the Lymph Nodes Authors: Jyh Liang Hor and Scott N. Mueller, date: 09/20/2016, view: 6623, Q&A: 0

Our protocol describes a simple method that allows tracking of dendritic cells (DC) migration from the flank skin to draining lymph nodes (LN) using a red fluorescent dye tetramethylrhodamine-5-isothiocyanate (TRITC). TRITC is a photostable dye that readily labels cells including DC in the skin and can survive repeated exposure of two-photon laser ...

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Isolation of Intestinal Mesenchymal Cells from Adult Mice Authors: Vasiliki Koliaraki and George Kollias, date: 09/20/2016, view: 15888, Q&A: 0

During the last 20 years intestinal mesenchymal cells (IMCs) have emerged as an important cell type that plays a central role in intestinal development and homeostasis, by providing both structural support and growth regulatory elements. IMCs also actively participate in wound healing responses, thus regulating pathologic conditions such as tissue ...

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Microbiology

Extraction and Quantification of GABA and Glutamate from Cyanobacterium Synechocystis sp. PCC 6803 Authors: Simab Kanwal and Aran Incharoensakdi, date: 09/20/2016, view: 8913, Q&A: 0

GABA (γ-amino butyric acid) is a biologically active four carbon non-protein amino acid found in prokaryotes and eukaryotes. Glutamate is a five carbon α-amino acid which can be converted to GABA catalyzed by the enzyme glutamate decarboxylase. In this protocol, we describe the procedure for extraction and quantification of GABA and glutamate from ...

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⁵⁹Fe Uptake Assays in Paracoccidioides Species Authors: Daniel J. Kosman, Elisa Flávia Luiz Cardoso Bailão, Mirelle Garcia Silva-Bailão and Célia Maria de Almeida Soares, date: 09/20/2016, view: 5174, Q&A: 0

Iron is an essential micronutrient required for virtually all organisms. This fact is related to the ability of the transition metal to exist in two oxidation states, the reduced ferrous (Fe²⁺) and the oxidized ferric (Fe³⁺). Given the relative availability of aqueous iron (the element which constitutes ~5% of the earth’s ...

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Purification and Identification of Novel Host-derived Factors for Influenza Virus Replication from Human Nuclear Extracts Authors: Kenji Sugiyama and Kyosuke Nagata, date: 09/20/2016, view: 7379, Q&A: 0

Recently, we identified two host cell-derived proteins as novel stimulatory factors of influenza virus RNA replication process, termed “Influenza virus REplication Factor-2 (IREF-2)”, from human nuclear extracts (NEs) by employing biochemical complementation assays (Sugiyama et al., 2015). Herein, we describe detailed methods for ...

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Neuroscience

Assessment of Mechanical Allodynia in Rats Using the Electronic Von Frey Test Authors: Jeremy Ferrier, Fabien Marchand and David Balayssac, date: 09/20/2016, view: 16696, Q&A: 0

Chronic pain is one of the most debilitating conditions, affecting one out of five people worldwide. Preclinical models in rodents represent a valuable tool for the study of pathophysiological mechanisms and the discovery of new analgesic drugs. However, pain evaluation in rodents is rather challenging. Altered response to mechanical or thermal ...

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In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling Authors: Ryohei Iwata and Takuji Iwasato, date: 09/20/2016, view: 6977, Q&A: 0

Dendritic spines are the post-synaptic structures that play a central role in excitatory synaptic transmission. Developmental spinogenesis relies on a variety of stimuli such as those derived from cell-cell communication and their downstream signaling. Here, we describe an in vitro assay of dendritic spine retraction using hippocampal ...

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Plant Science

Paper Roll Culture and Assessment of Maize Root Parameters Authors: Adel H. Abdel-Ghani, Darlene L. Sanchez, Bharath Kumar and Thomas Lubberstedt, date: 09/20/2016, view: 8961, Q&A: 0

Selection for genotypes with a vigorous root system could enhance the adaptation of maize under water and nutrient deficit soils. Although extensive genetic variation for root architecture has been reported (Kumar et al., 2012; Abdel-Ghani et al., 2014; Kumar et al., 2014; Pace et al., 2015), root traits have ...

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Extraction and Assays of ADP-glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains Authors: Konstantin Kulichikhin, Shalini Mukherjee and Belay T. Ayele, date: 09/20/2016, view: 9739, Q&A: 0

Starch biosynthesis in plants involves a network of enzymes of which adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 2.7.7.27), and soluble and granule bound starch synthases (SSS and GBSS, E.C. 2.4.1.21) play central roles. Here, we outline the protocol for extraction and assay of these enzymes in developing grains of ...

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Use of SCRI Renaissance 2200 (SR2200) as a Versatile Dye for Imaging of Developing Embryos, Whole Ovules, Pollen Tubes and Roots Authors: Thomas J. Musielak, Patrick Bürgel, Martina Kolb and Martin Bayer, date: 09/20/2016, view: 10745, Q&A: 1

Confocal laser scanning microscopy in combination with fluorescent proteins is a powerful tool for the study of sexual reproduction and other developmental processes in plants. In order to understand the origin and localization of fluorescent signals in a complex tissue, staining of cell outlines is often mandatory. Cell wall staining with SCRI ...

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