Volume: 7 Issue: 10 —BIO-PROTOCOL

Cancer Biology

Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method Authors: Pamela Bielli and Claudio Sette, date: 05/20/2017, view: 6668, Q&A: 0

RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, ...

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Semi-quantitative Analysis of H4K20me1 Levels in Living Cells Using Mintbody Authors: Yuko Sato and Hiroshi Kimura, date: 05/20/2017, view: 5938, Q&A: 0

Eukaryotic nuclear DNA wraps around histone proteins to form a nucleosome, a basic unit of chromatin. Posttranslational modification of histones plays an important role in gene regulation and chromosome duplication. Some modifications are quite stable to be an epigenetic memory, and others exhibit rapid turnover or fluctuate during the cell cycle. ...

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Nucleosome Positioning Assay Authors: Zhongliang Zhao and Holger Bierhoff, date: 05/20/2017, view: 7745, Q&A: 0

The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, ...

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Murine Bronchoalveolar Lavage Authors: Fan Sun, Gutian Xiao and Zhaoxia Qu, date: 05/20/2017, view: 16714, Q&A: 1

A basic Bronchoalveolar lavage (BAL) procedure in mouse is described here. Cells and fluids obtained from BAL can be analyzed by Hema3-staining, immunostaining, Fluorescence-activated cell sorting (FACS), PCR, bicinchoninic acid protein assay, enzyme-linked immunosorbent assay (ELISA), luminex assays, etc., to examine the immune cells, ...

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Isolation of Murine Alveolar Type II Epithelial Cells Authors: Fan Sun, Gutian Xiao and Zhaoxia Qu, date: 05/20/2017, view: 7317, Q&A: 0

We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative ...

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Imaging the Pharynx to Measure the Uptake of Doxorubicin in Caenorhabditis elegans Authors: Sivathevy Amirthagunabalasingam, Arturo Papaluca, Taramatti Harihar and Dindial Ramotar, date: 05/20/2017, view: 5803, Q&A: 0

Caenorhabditis elegans offers an array of advantages to investigate the roles of uptake transporters. Herein, an epifluorescent microscopy approach was developed to monitor the uptake of the autofluorescent anticancer drug, doxorubicin, into the pharynx of C. elegans by organic cation transporters.

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Cell Biology

Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines Authors: Daniela Boehm and Melanie Ott, date: 05/20/2017, view: 7652, Q&A: 0

The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral treatments with ...

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Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation Authors: Go R. Sato, Tatsuma Ban and Tomohiko Tamura, date: 05/20/2017, view: 9211, Q&A: 0

While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where ...

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Immunology

Lung Section Staining and Microscopy Authors: Xiaofeng Zhou and Bethany B Moore, date: 05/20/2017, view: 15260, Q&A: 0

Our protocol describes immunofluorescent staining, hematoxylin and eosin staining and Masson’s trichrome staining on lung sections.

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Detection of ASC Oligomerization by Western Blotting Authors: Jérôme Lugrin and Fabio Martinon , date: 05/20/2017, view: 11537, Q&A: 0

The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote ...

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Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi Authors: Noelia Lander, Miguel A. Chiurillo, Aníbal E. Vercesi and Roberto Docampo, date: 05/20/2017, view: 9566, Q&A: 0

To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then ...

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Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library Authors: Brooke A Napier and Denise M Monack, date: 05/20/2017, view: 8426, Q&A: 0

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of ...

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Microbiology

Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine Authors: Eric M. Velazquez, Fabian Rivera-Chávez and Andreas J. Bäumler, date: 05/20/2017, view: 4976, Q&A: 0

In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, ...

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Molecular Biology

Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line Authors: Stuart W. Tompson and Terri L. Young, date: 05/20/2017, view: 7469, Q&A: 1

There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator ...

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Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes Authors: Brendan A. Palmer, Zoya Dimitrova, Pavel Skums, Orla Crosbie, Elizabeth Kenny-Walsh and Liam J. Fanning, date: 05/20/2017, view: 3987, Q&A: 0

Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary ...

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CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae Authors: Yu Sasano and Satoshi Harashima, date: 05/20/2017, view: 6551, Q&A: 0

Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high ...

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A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism Author: Hiroshi Arakawa, date: 05/20/2017, view: 6352, Q&A: 0

While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in ...

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Neuroscience

Primary Olfactory Ensheathing Cell Culture from Human Olfactory Mucosa Specimen Authors: Mansoureh Hashemi and Mahmoudreza Hadjighassem, date: 05/20/2017, view: 5043, Q&A: 0

The human olfactory mucosa is located in the middle and superior turbinates, and the septum of nasal cavity. Olfactory mucosa plays an important role in detection of odours and it is also the only nervous tissue that is exposed to the external environment. This property leads to easy access to the olfactory mucosa for achieving various researches. ...

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Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining Authors: Chao Wang, Feng Yue and Shihuan Kuang, date: 05/20/2017, view: 15330, Q&A: 1

Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber ...

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A Tactile-visual Conditional Discrimination Task for Testing Spatial Working Memory in Rats Authors: Alicia Edsall, Zachary Gemzik and Amy Griffin, date: 05/20/2017, view: 4470, Q&A: 0

This protocol describes a novel dual task comparison across two variants of a tactile-visual conditional discrimination (CD) T-maze task, one is dependent upon spatial working memory (SWM; CDWM) and the other one (CD_STANDARD) is not. The task variants are equivalent in their sensory and motor requirements and overt behavior of the rat. ...

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Locomotor Assay in Drosophila melanogaster Authors: Qingqing Liu, Jingsong Tian, Xing Yang, Yan Li and Aike Guo, date: 05/20/2017, view: 6418, Q&A: 0

This protocol describes a simple locomotor assay in Drosophila melanogaster. In brief, the locomotor of each single fly in the culture dish is recorded by a web camera. The moving time, walking length, speed and the locomotor trails of the single fly could be quantitatively analyzed.

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Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice Authors: Julian Christopher Assmann, Kristin Müller, Jan Wenzel, Thomas Walther, Josefine Brands, Peter Thornton, Stuart M. Allan and Markus Schwaninger, date: 05/20/2017, view: 8580, Q&A: 0

Brain endothelial cells are the major building block of the blood-brain barrier. To study the role of brain endothelial cells in vitro, the isolation of primary cells is of critical value. Here, we describe a protocol in which vessel fragments are isolated from adult mice. After density centrifugation and mild digestion of the fragments, ...

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Plant Science

1-MCP (1-methylcyclopropene) Treatment Protocol for Fruit or Vegetables Authors: Gamrasni Dan, Goldway Martin, Stern Yosi, Breitel Dario and Aharoni Asaph, date: 05/20/2017, view: 10932, Q&A: 4

1-MCP (1-methylcyclopropene) is a simple synthetic hydrocarbon molecule that interacts with the ethylene receptor and inhibits the response of fruit or plant to ethylene. 1-MCP has opened new opportunities in handling harvested crops and serves as a powerful tool to learn about plant response to ethylene (Watkins and Miller, 2006). 1-MCP is ...

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Simple Spectroscopic Determination of Nitrate, Nitrite, and Ammonium in Arabidopsis thaliana Authors: Takushi Hachiya and Yuki Okamoto, date: 05/20/2017, view: 11970, Q&A: 1

Plants use nitrate, nitrite, and ammonium as inorganic nitrogen (N) sources. These N compounds are included in plant tissues at various concentrations depending on the balance between their uptake and assimilation. Thus, the contents of nitrate, nitrite, and ammonium are physiological indicators of plant N economy. Here, we describe a protocol for ...

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Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum Authors: Rémi Peyraud, Timothy P. Denny and Stéphane Genin, date: 05/20/2017, view: 5365, Q&A: 0

Soluble exopolysaccharide is a major virulence factor produced by the plant pathogen Ralstonia solanacearum. Its massive production during plant infection is associated with the arrest of water flow in xylem vessels leading eventually to plant death. The composition of this heavy macromolecule includes mainly N-acetylgalactosamine. Here ...

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Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen Authors: Ning Yang, Bing Han, Lingtong Liu, Hao Yang and Tai Wang, date: 05/20/2017, view: 5225, Q&A: 0

Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separates the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous ...

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Protein Isolation from Plasma Membrane, Digestion and Processing for Strong Cation Exchange Fractionation Authors: Ning Yang, Bing Han and Tai Wang, date: 05/20/2017, view: 6799, Q&A: 0

Plasma membrane (PM) proteins play crucial roles in diverse biological processes. But their low abundance, alkalinity and hydrophobicity make their isolation a difficult task. This protocol describes an efficient method for PM proteins isolation, digestion and fractionation so that they can be well prepared for mass spectrometry analysis.

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Stem Cell

Efficient Production of Functional Human NKT Cells from Induced Pluripotent Stem Cells − Reprogramming of Human Vα24⁺iNKT Cells Authors: Daisuke Yamada, Tomonori Iyoda, Kanako Shimizu, Yusuke Sato, Haruhiko Koseki and Shin-ichiro Fujii, date: 05/20/2017, view: 7736, Q&A: 0

Antigen-specific T cell-derived induced pluripotent stem cells (iPSCs) have been shown to re-differentiate into functional T cells and thus provide a potential source of T cells that could be useful for cancer immunotherapy. Human Vα24⁺ invariant natural killer T (Vα24⁺iNKT) cells are subset of T cells that are characterized ...

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Past Issues in 2017

Volume: 7 Issue: 10

Cancer Biology

Cell Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Plant Science

Stem Cell