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Past Issue in 2017
Volume: 7, Issue: 21
Biochemistry
Preparation of the Partially Methylated Alditol Acetates Derived from CS Tetrasaccharides Containing Galactose for the Gas Chromatography/Mass Spectrometry Analysis
Chondroitin sulfate (CS), a member of the glycosaminoglycan (GAG) family of carbohydrates, is composed of linear, sulfated repeating disaccharide sequences of N-acetyl-D-galactosamine (GalNAc) and glucuronic acid (GlcA). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S(β1-3)Galactose(β1-]-branched CS-E was identified from the clam species M. chinensis. This protocol details a methodology to analyze the glycosidic linkages of galactose in KS disaccharide-branched CS by GC-MS analysis. A complementary method for the identification and characterization of KS-branched CS in M. chinensis can be found in Higashi et al. (2016).
Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography.
Cancer Biology
In vitro NLK Kinase Assay
This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.
Xenograft Mouse Model of Human Uveal Melanoma
Uveal melanoma (UM) is a malignant intraocular tumor in adults. Metastasis develops in almost half of the patients and over 90% of the metastases are in the liver. With the advances in molecular targeting therapy for melanoma, a proper metastasis animal model is of increasing importance for testing the accuracy and effectiveness of systemic therapies. Here, we describe a xenograft model for mimicking human UM liver metastasis by injecting human UM cells into the vitreous cavity in nude mice. The athymic nude mice are immunocompromised and suitable for xenograft tumor growth and metastasis, and intravitreal injection of cells is a quicker and easier operation under a binocular scope, thereby it is simple and effective to test human UM growth and metastasis.
Cell Biology
Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues
Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy related pathological conditions like preterm premature rupture of membranes (PPROM), collagen of the fetal membrane undergoes dynamic remodeling defining biochemical properties of the fetal membrane. The protocol in this article describes the histochemical method to stain total collagen by Picrosirius red stain which is a simple, quick and reliable method. This protocol can be used on paraformaldehyde (PFA) and formaldehyde fixed paraffin embedded tissue sections. We further describe the staining and distribution of collagen in different mouse reproductive tissues and also demonstrate how this technique in combination with polarization microscopy is useful to detect the distribution of different subtypes of collagen.
A Bioreactor Method to Generate High-titer, Genetically Stable, Clinical-isolate Human Cytomegalovirus
Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in transplant patients and a leading cause of congenital birth defects (Saint Louis, 2016). Vaccination and therapeutic studies often require scalable cell culture production of wild type virus, represented by clinical isolates. Obtaining sufficient stocks of wild-type clinical HCMV is often labor intensive and inefficient due to low yield and genetic loss, presenting a barrier to studies of clinical isolates. Here we report a bioreactor method based on continuous infection, where retinal pigment epithelial (ARPE-19) cells adhered to microcarrier beads are infected in a bioreactor and used to produce high-titers of clinical isolate HCMV that maintain genetic integrity of key viral tropism factors and the viral genome. In this bioreactor, an end-stage infection can be maintained by regular addition of uninfected ARPE-19 cells, providing convenient preparation of 107-108 pfu/ml of concentrated TB40/E IE2-EYFP stocks without daily cell passaging or trypsinization. Overall, this represents a 100-fold increase in gain of virus production of 100-times compared to conventional static-culture plates, while requiring 90% less handling time. Moreover, this continuous infection environment has the potential to monitor infection dynamics with applications for real-time tracking of viral evolution.
Nematode Epicuticle Visualisation by PeakForce Tapping Atomic Force Microscopy
The free-living soil nematode Caenorhabditis elegans has become an iconic experimental model animal in biology. This transparent animal can be easily imaged using optical microscopy to visualise its organs, tissues, single cells and subcellular events. The epicuticle of C. elegans nematodes has been studied at nanoscale using transmission and scanning (SEM) electron microscopies. As a result, imaging artefacts can appear due to embedding the worms into resins or coating the worms with a conductive gold layer. In addition, fixation and contrasting may also damage the cuticle. Conventional tapping mode atomic force microscopy (AFM) can be applied to image the cuticle of the dried nematodes in air, however this approach also suffers from imaging defects. Ideally, the nematodes should be imaged under conditions resembling their natural environment. Recently, we reported the use of PeakForce Tapping AFM mode for the successful visualisation and numerical characterisation of C. elegans nematode cuticle both in air and in liquid (Fakhrullina et al., 2017). We imaged the principal nematode surface structures and characterised mechanical properties of the cuticle. This protocol provides the detailed description of AFM imaging of liquid-immersed C. elegans nematodes using PeakForce Tapping atomic force microscopy.
Immunogold Localization of Molecular Constituents Associated with Basal Bodies, Flagella, and Extracellular Matrices in Male Gametes of Land Plants
Male gametes (spermatozoids) are the only motile cells produced during the life cycle of land plants. While absent from flowering and most cone-bearing plants, motile cells are found in less derived taxa, including bryophytes (mosses, liverworts and hornworts), pteridophytes (lycophytes and ferns) and some seed plants (Ginkgo and cycads). During development, these cells undergo profound changes that involve the production of a locomotory apparatus, unique microtubule (MT) arrays, and a series of special cell walls that are produced in sequence and are synchronized with cellular differentiation. Immunogold labeling in the transmission electron microscope (TEM) provides information on the exact location and potential function of macromolecules involved with this developmental process. Specifically, it is possible to localize epitopes to proteins that are associated with the cellular inclusions involved in MT production and function. Spermatogenesis in these plants is also ideal for examining the differential expression of carbohydrates and glycoproteins that comprise the extracellular matrixes associated with the dramatic architectural changes in gamete shape and locomotory apparatus development. Here we provide methodologies using monoclonal antibodies (MAbs) and immunogold labeling in the TEM to localize macromolecules that are integral to spermatozoid development.
Immunology
Proximal Ligation Assay (PLA) on Lung Tissue and Cultured Macrophages to Demonstrate Protein-protein Interaction
In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have complementary oligonucleotide moieties attached to them. If the two antigens are in close proximity (presumably interacting with each other), the complementary oligonucleotides can anneal and fluorescent nucleotides can be incorporated in a single DNA polymerization step. Under a microscope, these reactions appear as punctate fluorescent spots, indicating successful PLA reaction and suggesting protein-protein interaction between the two antigens.
Microbiology
Isolation of Rice Stripe Virus Preparation from Viruliferous Small Brown Planthoppers and Mechanic Inoculation on Rice
Tenuiviruses can infect the plants of the family Poaceae, and cause serious loss of crops, particularly rice and maize, in South-Eastern Asian countries. Tenuiviruses usually depend on insect vectors for their transmission and cannot be transmitted between plants through wounds or abrasions. Rice stripe virus (RSV), a typical member of tenuiviruses, is efficiently transmitted by the small brown planthopper Laodelphax striatellus in a persistent-propagative manner to cause rice stripe disease. Here we presented a convenient method, the midrib micro-injection, to mechanically inoculate insect-derived RSV into rice leaves for conducting pathogenicity assay on rice plants.
Neuroscience
Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells
There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using two different culture systems to assess their potential regarding proliferation and differentiation in a reductionist model. While the neurosphere assay is primarily performed to directly study the proliferative and differentiation potential of cells in individual brains, the monolayer culture allows single cell analysis in a rather homogeneous cell population. Here, we describe the isolation, culturing methods and differentiation of neural precursor cells in both systems.
Assessment of Aversion of Acute Pain Stimulus through Conditioned Place Aversion
Pain is a complex experience. The aversive component of pain has been assessed through conditioned place aversion in rodents. However, this behavioral test does not allow the evaluation of the aversion of an acute pain stimulus. In Zhang et al. (2017), we provide an updated version of a Conditioned Place Aversion paradigm to address this challenge. In this protocol, a detailed version of this method is described.
Sensitive Estimation of Flavor Preferences in STFP Using Cumulative Time Profiles
Social transmission of food preference (STFP) is observed among rodents between a demonstrator and a naïve hungry observer. During social interaction, hungry observer receives information about safety of the food consumed by the demonstrator. This task has been implemented to develop a single trial non-aversive learning task in order to test hippocampus dependent non-spatial memory in rodents. In this protocol, we describe some novel modifications to the conventional STFP protocol and analysis for more sensitive estimation of change in preferences. Using this method, preference trends can be observed for weeks after training, allowing one to probe the role of systems consolidation (SC) in declarative memory that is relatively independent of spatial navigation.
Stem Cell
Isolation of Primary Human Skeletal Muscle Cells
Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study.