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Past Issue in 2016
Volume: 6, Issue: 18
Immunology
Isolation of Intestinal Mesenchymal Cells from Adult Mice
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 repair, inflammation, fibrosis and carcinogenesis (Powell et al., 2011). We have recently demonstrated that intestinal mesenchymal-specific signals play important in vivo physiological roles in intestinal inflammation and carcinogenesis (Koliaraki et al., 2012; Roulis et al., 2014; Koliaraki et al., 2015). Here we describe the enzymatic method used for the isolation and culture of mesenchymal cells from the adult mouse intestine.
Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia
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 the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.
Imaging Thick Lymph Node Tissue Sections
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 that is typically under-represented in thin sections (10-20 μm) used for conventional confocal microscopy. Application of 2-photon microscopy facilitates imaging through the thick volume of the vibratome sections. In combination with automated image processing software, a thick lymph node cross-section image also facilitates quantitation of cellular events within a relatively large area of the tissue, thus providing a clearer picture on the spatial distribution of cellular events of interest (e.g., T cell clustering). This method can also readily be applied to other tissues, such as the spleen or skin.
Peptide Loading on MHC Class I Molecules of Tumor Cells
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. This protocol allows for detachment of the endogenous peptides bound to MHC class I molecules by preserving them for the binding of high affinity synthetic peptides. The complete dissociation of endogenous peptides by mild acid treatment as well as the binding of synthetic peptides to MHC class I molecules will be evaluated measuring HLA class I molecules express on the cell surface by flow cytometry. The mouse antibody W6/32 which recognizes β2m associated HLA-A, -B, -C, -E and -G heavy chains is suitable for this propose. Any tumor cell line that expresses surface HLA class I molecules is suitable for the assay. Another important aspect is to know the HLA class I typing of tumor cell line to allow selection of the known high affinity peptides.
Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents
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). Recently, we showed that protein extracts from gut commensal microbiota can activate retina-specific T cells, allowing these autoreactive T cells to then break through the blood-retinal barrier and trigger autoimmune uveitis in the recipient (Horai et al., 2015). The protocol below describes the method to prepare intestinal protein-rich extracts that can be used in various in vitro and in vivo immunological studies.
Skin TRITC Painting to Track Dendritic Cells Migrating to the Lymph Nodes
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 excitation for prolonged imaging duration. This method can be combined with various fluorescent antibody labels or transgenic mouse strains (such as CD11c-EYFP) to visualize distinct DC populations simultaneously.
Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells
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 activating signals transmitted by activating receptors interacting with specific ligands, leads to NK cell activation (Lanier, 2005; Raulet et al., 2001). Thus, the balance of the ligands expressed for inhibitory and activating receptors determines whether NK cells will become activated to kill the target cells. This protocol allows to assign a precise ligand specificity to any given receptor on NK cells. Thus, if a tumor cell expresses the ligand, this protocol will allow to evaluate its interaction with the specific receptor. In particular, killer cell immunoglobulin (Ig)-like receptors (KIR) recognize their ligands (HLA class I molecules) through the direct contact with HLA class I heavy chain residues and amino acid residues of the bound peptide. This protocol will allow to test the effect of amino acid substitutions or other mutations on the binding of KIR to HLA class I. We used this protocol to depict the role of ERAP1, a key component of the MHC class I antigen processing, in regulating NK cell function by controlling the engagement of inhibitory receptors (Cifaldi et al., 2015).
Microbiology
Extraction and Quantification of GABA and Glutamate from Cyanobacterium Synechocystis sp. PCC 6803
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 cells of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Apart from Synechocystis, this protocol has already been successfully tested for the cyanobacterium Nostoc punctiforme and for the green alga Tetraspora sp. CU2551. The protocol can also be used for the analyses of other primary amino acids. We have successfully employed this protocol in our studies of GABA and glutamate analyses in Synechocystis (Kanwal and Incharoensakdi, 2016).
Purification and Identification of Novel Host-derived Factors for Influenza Virus Replication from Human Nuclear Extracts
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 successive procedures for identification and purification of IREF-2, including large-scale suspension culture of HeLa S3 cells, preparation of NEs and separation of IREF-2 by sequential column chromatography steps. This protocol can be modified and used for purification and identification of the other unknown nuclear protein(s) of your interest.
59Fe Uptake Assays in Paracoccidioides Species
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 crust) one is not surprised that iron is the most common prosthetic element in biology. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA) (Kosman, 2013). In this way, the uptake of iron in the absence or presence of the reducing agent ascorbic acid can be investigated by 59Fe uptake assays, as previously described (Eide et al., 1992). In the presence of ascorbic acid, the reductive-independent 59Fe uptake route is investigated. On the other hand, in the absence of ascorbic acid, the reductive-dependent 59Fe uptake route is stimulated. Using this strategy for the human pathogenic fungus Paracoccidioides species, the results showed that iron uptake by Pb01 in the absence of ascorbic acid was low, unlike what was observed for Pb18. These results suggest that only in Pb18 the iron uptake pathway is coupled to a ferric reductase (Bailão et al., 2015). In this protocol, we describe how to perform 59Fe uptake assays in Paracoccidioides species.
Neuroscience
Assessment of Mechanical Allodynia in Rats Using the Electronic Von Frey Test
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 stimuli is commonly used as behavioral outcome to measure pain sensitivity. This protocol introduces a method for assessing static mechanical pain hypersensitivity in rats using an electronic von Frey (VF) test. The electronic VF is an evolution of the manual VF hairs previously described by Kim and Chung (1991). In this previous procedure, 6 calibrated nylon filaments (diameters of 0.13, 0.23, 0.31, 0.48, 0.52, and 0.59 mm, respectively) are perpendicularly applied to the plantar surface of the rat hind paw, to deliver the following defined pressures: 5.89, 9.81, 27.0, 74.4, 124, and 205 mN. Each application has to be repeated several times for each filament to determine the mechanical threshold. Comparatively, the electronic VF is relatively easy-to-use and particularly suited for pharmacological studies with precise time-points. The electronic VF can be used as a behavioural read-out for a wide range of models, including inflammatory and neuropathic pain. The following protocol was originally published in Ferrier et al. (2013), Grégoire et al. (2014) and in Ferrier et al. (2015).
In vitro Assay for Dendritic Spine Retraction of Hippocampal Neurons with Sparse Labeling
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 slice culture, in which individual neurons are sparsely and brightly labeled by the Supernova method, for the study of molecular mechanisms of spine development.
Plant Science
Use of SCRI Renaissance 2200 (SR2200) as a Versatile Dye for Imaging of Developing Embryos, Whole Ovules, Pollen Tubes and Roots
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 Renaissance 2200 (SR2200) has recently been described as a method of choice to study plant reproductive processes (Musielak et al., 2015). In this protocol, we present detailed instructions on the use of SR2200 to stain cell walls in different Arabidopsis tissues.
Extraction and Assays of ADP-glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains
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 wheat (Triticum aestivum L.). The principle of the assays outlined is based on a coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be measured easily by spectrophotometer. This protocol does not need expensive labelled chemicals and can be carried out using equipment commonly found in a biochemical laboratory. We applied this protocol to study the dynamics of AGPase, SSS and GBSS activity in developing wheat grains at different time points after anthesis.
Paper Roll Culture and Assessment of Maize Root Parameters
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 been seldom considered as selection criteria to improve yield in maize, mainly because characterization of root morphology in the field is laborious, inaccurate and time consuming (Tuberosa and Salvi, 2007). Characterization of root traits under hydroponic conditions in this case has the advantage of screening a high number of genotypes in a small space (in a growth chamber) within a short period of time (2-3 weeks). Thus, it saves the time and effort required for screening maize genotypes with vigorous root systems and might be helpful to monitor root development at different growth stages.