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Past Issue in 2016
Volume: 6, Issue: 21
Biochemistry
Quantitation of Cytochromes b559, b6, and f, and the Core Component of Photosystem I P700 in Cyanobacterial Cells
Cytochrome (Cyt) b559, an important and essential core component of photosystem II in the photosynthetic electron transport system, is a heme-bridged heterodimer protein composed of an alpha subunit (PsbE) and a beta subunit (PsbE), and its reduced form has an absorption maximum in the α-band at 559 nm. The amounts of Cyt b559 can be determined by spectrophotometrical measurement of reduced minus oxidized difference spectra that are normalized with absorbance of isosbestic point at 580 nm. The authors use differential extinction coefficients of Cyt b559 [Δε(559-580 nm) = 15.5 mM-1·cm-1], which have been reported by Garewal and Wasserman (1974). In addition to the Cyt b559, this procedure can be used for quantitation of Cyt b6 and Cyt f, the subunits of the Cyt b6/f complex, and P700, one of the core components of photosystem I. This protocol, which is adapted from Fujita and Murakami (1987), is used in a cyanobacterium, Synechococcus elongatus PCC 7942, and also in other cyanobacterial strains including Synechocystis sp. PCC 6803.
Cancer Biology
Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation
The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation.The protocol can be divided into four main steps: preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement. This assay is limited to manual or semiautomatic screening, and can be used in an efficient and sensitive manner to test chemotherapeutic drugs or small molecules in adherent cells. It also has applications in evaluating the effects of gene expression modulation (knockdown, gene expression upregulation), as well as to study the effects of miRNA replacement on cell proliferation (Kasinski et al., 2015).
Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs
This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTrackerTM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than necroptosis.
Fat Turnover Assay in Drosophila
Like all animals, Drosophila shows robust fat (triglyceride) turnover, i.e., they synthesize, store and utilize triglyceride for their daily metabolic needs. The protocol describes a simple assay to measure this turnover of triglycerides in Drosophila.
Cell Biology
Determination of the Glycolysis and Lipogenesis in Culture of Hepatocytes
Metabolic flux analyses are needed to provide insights into metabolic regulation that occurs in cells. The current protocol describes fast and reproducible methods for determining glycolysis and de novo lipogenesis of hepatocytes. Primary culture of hepatocytes is an ‘in vitro’ model useful to study liver glucose and lipid metabolism (Denechaud et al., 2016). The protocol is divided in 2 parts. Part I: Glycolysis experiment is assessed using the Seahorse extracellular flux (XF) analyser. Glycolysis is determined via the measurement of the extracellular acidification rate (ECAR) of the media, which come predominately from the cellular excretion of lactic acid after the conversion of glucose in pyruvate. Part II: De novo lipogenesis experiment determines the radioactive C14 incorporation in triglycerides (TG) from acetate C14 precursor. After 2 h acetate supplementation to the media lipids are extracted and separated by TLC (Thin Layer Chromatography) prior quantification of newly synthetized TG labelled.
Immunology
Reconstruction of the Mouse Inflammasome System in HEK293T Cells
The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to microbes and/or danger signals, the inflammasome assembles and serves as a platform for the activation of caspase-1. Caspase-1 activation promotes the processing and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β), IL-18, and IL-33 as well as pyroptosis induction (Gross et al., 2011; Arend et al., 2008), which elicit inflammatory responses. Here, we describe how to co-transfect the NLRP3 inflammasome components into HEK293T cells, which enables inflammasome activation and the production of IL-1β upon stimulation with nigericin.
Microbiology
Sequencing of Ebola Virus Genomes Using Nanopore Technology
Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation sequencing methods, and was successfully used under outbreak conditions (Hoenen et al., 2016; Quick et al., 2016). Here we describe a protocol used for sequencing of Ebola virus under outbreak conditions using Nanopore technology, which we successfully implemented at the CDC/NIH diagnostic laboratory (de Wit et al., 2016) located at the ELWA-3 Ebola virus Treatment Unit in Monrovia, Liberia, during the recent Ebola virus outbreak in West Africa.
Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography
The stringent response in bacteria is a stress response that is mediated by the signaling molecules guanosine tetraphosphate and pentaphosphate [(p)ppGpp], alarmones that are also directly related to virulence. Therefore, determination of (p)ppGpp levels is crucial for studying the stringent response. The protocol here outlines in a step-wise manner the detection of (p)ppGpp in the bacterium Streptomyces coelicolor during stringent response (Strauch et al., 1991) by thin layer chromatography (TLC). In the example shown here, stringent response is induced by addition of serine hydroxamate, an inhibitor of seryl tRNA synthetase. This protocol was first published in Molecular Microbiology (Sivapragasam and Grove, 2016).
Molecular Biology
Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation
dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses.
Neuroscience
Protocol for Primary Microglial Culture Preparation
Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse pups. The overall steps include brain cell dissociation, mixed glial cell culture, and microglia isolation.
Microglial Phagocytosis Assay
Phagocytosis is essential for microglial clearance of apoptotic cells, extracellular protein aggregates, and infectious bacteria in the central nervous system (CNS). While the preparation of primary microglial culture has been described elsewhere, this protocol describes the microglial phagocytosis experimental procedure and the subsequent measurement of microglial phagocytic ability using fluorescent latex beads or fluorescent amyloid beta 42 (Aβ42) peptides.
Apparatus and General Methods for Exposing Rats to Audiogenic Stress
Most organisms react innately to the sudden onset of environmental stimulation. Audiogenic or loud noise in rodents provides an effective threatening signal to study the central nervous circuits responsible for the elaboration of various responses typically elicited by threatening/stressful environmental stimulation. Audiogenic stress offers many advantages over other environmental stimulation, including exquisite control over timing, intensity, and frequency, using off-the-shelf components that produce easily reproducible results. This protocol provides blueprints for the construction of sound attenuation chambers, the associated sound generation, amplification, and delivery equipment, and general procedures sufficient to elicit multimodal responses to loud noises in rodents.
Plant Science
Hydrogen Peroxide Measurement in Arabidopsis Root Tissue Using Amplex Red
This protocol describes the measurement of hydrogen peroxide (H2O2) content in Arabidopsis root tissue by using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit. When root tissue is disrupted and resuspended in phosphate buffer, H2O2 is released from the cells. The obtained root extracts containing H2O2 can be mixed with a solution containing Amplex® Red reagent (10-acetyl-3,7-dihydrophenoxazine). In the presence of horseradish peroxidase, the Amplex® Red reagent reacts with H2O2 in a 1:1 stoichiometry. The resulting product is the red-fluorescent compound resorufin which can be detected fluorometrically or spectrophotometrically. Our protocol is based on the manual of the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit and describes a step-by-step procedure with a detailed description of the necessary controls and data analysis. We have also included modifications of the protocol, notes and examples that intend to aid the user in easily reproducing the assay with their own samples.
A Ribosome Footprinting Protocol for Plants
Ribosome footprinting, or Ribo-seq, has revolutionized the studies of translation. It was originally developed for yeast and mammalian cells in culture (Ingolia et al., 2009). Herein, we describe a plant-optimized hands-on ribosome footprinting protocol derived from previously published procedures of polysome isolation (Ingolia et al., 2009; Mustroph et al., 2009) and ribosome footprinting (Ingolia et al., 2009; Ingolia et al., 2013). With this protocol, we have been able to successfully isolate and analyze high-quality ribosomal footprints from different stages of in vitro grown Arabidopsis thaliana plants (dark-grown seedlings [Merchante et al., 2015] and 13-day-old plantlets in plates and plants grown in liquid culture [unpublished results]).
Metabolite Profiling of Mature Arabidopsis thaliana Seeds Using Gas Chromatography-Mass Spectrometry (GC-MS)
Metabolite profiling using gas chromatography-mass spectrometry (GC-MS) permits the annotation and quantification of a relatively wide variety of metabolites, covering a wide range of biochemical groups of metabolites. Lisec et al. (2006) established a method for GC-MS profiling in plants. Based on this protocol, we provide here a detailed GC-MS-based metabolite profiling protocol to identify compounds belonging to several biochemical groups in the primary metabolism of mature Arabidopsis thaliana seeds (Cohen et al., 2014). The protocol uses methoxyamine hydrochloride and N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) as derivatization reagents, as previous studies indicated these are the most appropriate compounds for profiling of plant metabolites. The protocol is relatively rapid, delivers reproducible results, and can be employed to profile metabolites of many other types of plant tissues with only minor modifications. In this context, developing seeds can serve as an excellent system for studying metabolic regulation, since during their development, a massive synthesis of reserve compounds occurs controlled under tight transcriptional regulation and associated with temporally distinct metabolic switches.
Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment
This protocol can be used to inhibit the biosynthesis of polyamines, specifically putrescine, in tomato plants grown with NH4+ as a solely N source. In general, polyamines are positively charged small metabolites implicated in physiological processes, including organogenesis, embryogenesis, floral initiation and development, leaf senescence, pollen tube growth, fruit development and ripening and participate in the response to abiotic and biotic stresses (Tiburcio et al., 2014). Polyamines are synthesized from amino acids by decarboxylation of ornithine or arginine by ornithine decarboxylase (ODC) or arginine decarboxylase (ADC), respectively (Walters, 2003). Tomato plants grown with NH4+ as the sole N source presented an increase of putrescine content in leaves (Fernández-Crespo et al., 2015). To assess the importance of putrescine accumulation, DL-α-(Difluoromethyl)arginine (DFMA) and DL-α-(Difluoromethyl)ornithine (DFMO), inhibitors of putrescine synthesis, were used as irreversible inhibitors of ADC and ODC enzymes, respectively (Fallon and Phillips, 1988), with the purpose of reducing cellular putrescine accumulation induced by NH4+ nutrition. The inhibitor solution containing 2 mM DFMA and 5 mM DFMO was applied directly to each pot during the week prior to sample collection. Putrescine content was reduced by 35.3% in tomato plants grown with NH4+.
Determination of Recombinant Mannitol-1-phosphate Dehydrogenase Activity from Ectocarpus sp.
Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of the recombinant catalytic domain of one of the three M1PDHs identified in Ectocarpus sp. This recombinant catalytic domain, named hereafter M1PDHcat, catalyzes the reversible conversion of fructose-6-phosphate (F6P) to mannitol-1-phosphate (M1P) using NAD(H) as a cofactor. M1PDHcat activity was assayed in both directions i.e., F6P reduction and M1P oxidation (Figure 1).Figure 1. Reversible reaction of mannitol-1-phosphate dehydrogenase
PEA-CLARITY: Three Dimensional (3D) Molecular Imaging of Whole Plant Organs
Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material. Therefore, this protocol facilitates protein localisation of intact tissue in 3D whilst retaining cellular structure.
Cytohistological Analyses of Mega-sporogenesis and Gametogenesis in Ovules of Limonium spp.
Limonium spp. are known to have sexual and apomixis (asexual reproduction through seeds) reproductive modes. Here, we present dissection protocol developed for ovules of Limonium spp. using differential interference contrast (DIC) microscopy. This protocol permits better handling of ovules and offers certain advantages over earlier techniques particularly in larger ovules. This method also enables observation of meiosis and embryo sac development in intact ovules, and the ready detection of events distinguishing sexual and apomictic development.
Stem Cell
Allogeneic Transplantation of Testicular Hyperplasia in rag1 Mutant Zebrafish
Allogeneic organ transplantation is a powerful tool for clinical and basic research studies. However, the graft is often rejected by the host organism. Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish. These zebrafish escaped rejection, which made it possible to successfully transplant fragments of an allogeneic testis and testicular hyperplasia. This protocol can be used to amplify and maintain testicular hyperplasia grafts for several years (Kawasaki et al., 2016). The amplified hyperplasias are likely to be a good source of somatic and germ cells such as Sertoli cells and spermatogonial stem cells.