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Past Issue in 2017
Volume: 7, Issue: 1
Biochemistry
Measuring Oxidative Stress in Caenorhabditis elegans: Paraquat and Juglone Sensitivity Assays
Oxidative stress has been proposed to be one of the main causes of aging and has been implicated in the pathogenesis of many diseases. Sensitivity to oxidative stress can be measured by quantifying survival following exposure to a reactive oxygen species (ROS)-generating compound such as paraquat or juglone. Sensitivity to oxidative stress is a balance between basal levels of ROS, the ability to detoxify ROS, and the ability to repair ROS-mediated damage.
Examination of the Interaction between a Membrane Active Peptide and Artificial Bilayers by Dual Polarisation Interferometry
Examining the interaction of peptides with lipid bilayers to determine binding kinetics is often performed using surface plasmon resonance (SPR). Here we describe the technique of dual polarisation interferometry (DPI) that provides not only information on the kinetics of the peptide binding to the bilayer, but also how the peptide affects the lipid order of the bilayer.
Cancer Biology
In vivo Efficacy Studies in Cell Line and Patient-derived Xenograft Mouse Models
In vivo xenograft models derived from human cancer cells have been a gold standard for evaluating the genetic drivers of cancer and are valuable preclinical models for evaluating the efficacy of cancer therapeutics. Recently, patient-derived tumorgrafts from multiple tumor types have been developed and shown to more accurately recapitulate the molecular and histological heterogeneity of cancer. Here we detail the procedures for developing patient-derived xenograft models from breast cancer tissue, cell-based xenograft models, serial tumor transplantation, tumor measurement, and drug treatment.
Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro
Hydrogel systems composed of purified extracellular matrix (ECM) components (such as collagen, fibrin, Matrigel, and methylcellulose) are a mainstay of cell and molecular biology research. They are used extensively in many applications including tissue regeneration platforms, studying organ development, and pathological disease models such as cancer. Both the biochemical and biomechanical properties influence cellular and tissue compatibility, and these properties are altered in pathological disease progression (Cox and Erler, 2011; Bonnans et al., 2014). The use of cell-embedded hydrogels in disease models such as cancer, allow the interrogation of cell-induced changes in the biomechanics of the microenvironment (Madsen et al., 2015). Here we report a simple method to measure these cell-induced changes in vitro using a controlled strain rotational rheometer.
Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction
Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.
In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease
Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic clipping of histone tails is continuously attracting interest of researchers in the field of chromatin biology. We can recapitulate H3-clipping by performing in vitro H3 cleavage assay. Here, we are presenting the detailed protocol to perform in vitro H3 cleavage assay.
Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing
The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded in a stroma formed primarily by fibroblasts that produce an extracellular matrix and enwoven with blood vessels. The 3D mixed cell type spheroid model described here incorporates these key features of the tissue microenvironment that in vivo tumours exist in; namely the three-dimensional organisation, the most abundant stromal cell types (fibroblasts and endothelial cells), and extracellular matrix. This method combined with confocal microscopy can be a powerful tool to carry out drug sensitivity, angiogenesis and cell migration/invasion assays of different tumour types.
Immunology
Simultaneous Intranasal/Intravascular Antibody Labeling of CD4+ T Cells in Mouse Lungs
CD4+ T cell responses have been shown to be protective in many respiratory virus infections. In the respiratory tract, CD4+ T cells include cells in the airway and parenchyma and cells adhering to the pulmonary vasculature. Here we discuss in detail the methods that are useful for characterizing CD4+ T cells in different anatomic locations in mouse lungs.
FICZ Exposure and Viral Infection in Mice
The aryl hydrocarbon receptor (AHR) is known as a sensor for dioxins that mediates their toxicity, and also has important biophysiological roles such as circadian rhythms, cell differentiation and immune responses. 6-formylindolo(3,2-b)carbazole (FICZ), which is derived through the metabolism of L-tryptophan by ultraviolet B irradiation, is one of putative physiological ligands for AHR (Smirnova et al., 2016). It has recently been shown that endogenously-activated AHR signaling modulates innate immune response during viral infection (Yamada et al., 2016). This section describes how to treat mice with FICZ and to infect them with virus.
In vitro Treatment of Mouse and Human Cells with Endogenous Ligands for Activation of the Aryl Hydrocarbon Receptor
Activation of the aryl hydrocarbon receptor (AHR) by endogenous ligands has been implicated in a variety of physiological processes such as cell cycle regulation, cell differentiation and immune responses. It is reported that tryptophan metabolites, such as kynurenine (Kyn) and 6-formylindolo(3,2-b)carbazole (FICZ), are endogenous ligands for AHR (Stockinger et al., 2014). This protocol is designed for treatment with Kyn or FICZ in mouse embryonic fibroblasts (MEFs) or primary peripheral monocytes.
Microbiology
Bacterial Intracellular Sodium Ion Measurement using CoroNa Green
The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H+ and Na+ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na+ concentrations in living Escherichia coli cells using a sodium-sensitive fluorescent dye, CoroNa Green (Minamino et al., 2016). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na+ concentration quantitatively.
Measurements of Free-swimming Speed of Motile Salmonella Cells in Liquid Media
Bacteria such as Escherichia coli and Salmonella enterica swim in liquid media using the bacterial flagella. The flagellum consists of the basal body (rotary motor), the hook (universal joint) and the filament (helical screw). Since mutants with a defect in flagellar assembly and function cannot swim smoothly, motility assay is an easy way to characterize flagellar mutants. Here, we describe how to measure free-swimming speeds of Salmonella motile cells in liquid media. Free-swimming behavior under a microscope shows a significant variation among bacterial cells.
Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis
One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular reporter gene lacZ, encoding the enzyme β-galactosidase. We provide here a protocol for the in situ localization of β-galactosidase activity in cyanobacterial cells. This allows the same strain to be used for both a simple, quantitative, colorimetric assay with the substrate ortho-nitrophenyl-β-galactoside (ONPG) and for sensitive, fluorescence-based, cell-type localization of gene expression using 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG).
Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes
Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions within the column differ from ambient conditions. Here we present a pilot-scale column design and sampling protocol that is optimized for long-term spatial and temporal sampling. We utilized this experimental set-up over approximately 2 years to study a biologically active system designed to precipitate zinc-sulfides during sulfate reducing conditions; however, it can be adapted for the study of many flow-through systems where geochemical and/or molecular microbial analyses are desired. Importantly, these columns utilize retrievable solid-phase bags in conjunction with anoxic microbial techniques to harvest substrate samples while minimally disrupting column operation.
Neuroscience
Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn
Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing cellular disease models, such as pain and itch. Because of the specific features of SDHN, it is necessary to establish a reliable culture method that is suitable for testing neural response to various external stimuli in vitro.
Optogenetic Mapping of Synaptic Connections in Mouse Brain Slices to Define the Functional Connectome of Identified Neuronal Populations
Functional connectivity in a neural circuit is determined by the strength, incidence, and neurotransmitter nature of its connections (Chuhma, 2015). Using optogenetics the functional synaptic connections between an identified population of neurons and defined postsynaptic target neurons may be measured systematically in order to determine the functional connectome of that identified population. Here we describe the experimental protocol used to investigate the excitatory functional connectome of ventral midbrain dopamine neurons, mediated by glutamate cotransmission (Mingote et al., 2015). Dopamine neurons are made light sensitive by injecting an adeno-associated virus (AAV) encoding channelrhodopsin (ChR2) into the ventral midbrain of DATIREScre mice. The efficacy and specificity of ChR2 expression in dopamine neurons is verified by immunofluorescence for the dopamine-synthetic enzyme tyrosine hydroxylase. Then, slice patch-clamp recordings are made from neurons in regions recipient to dopamine neuron projections and the incidence and strength of excitatory connections determined. The summary of the incidence and strength of connections in all regions recipient to dopamine neuron projections constitute the functional connectome.
Plant Science
Dot Blot Analysis of N6-methyladenosine RNA Modification Levels
N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al., 2014; Shen et al., 2016). Here we describe the method for fast detection of total m6A levels in mRNA by dot blot analysis using a specific m6A antibody.
Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts
Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [45Ca] and [54Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [45Ca] or [54Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [45Ca] or [54Mn] can be determined by liquid scintillation counting and the relative distribution calculated.
In vitro Ubiquitin Dimer Formation Assay
The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can also be determined.
Stem Cell
Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors
Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016).
Ex vivo Culture of Adult Mouse Antral Glands
The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker’s protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016).