Volume: 8 Issue: 21 —BIO-PROTOCOL

Cancer Biology

Protocol for in situ Proximity Ligation Assay (PLA) and Microscopy Analysis of Epidermal Growth Factor Receptor (EGFR) Homodimerization Authors: Keiichi Ota and Taishi Harada, date: 11/05/2018, view: 6618, Q&A: 0

Oncogenic drivers play central roles in tumorigenesis as well as in tumor cell survival and proliferation. Mutations of the epidermal growth factor receptor gene (EGFR) that result in constitutive activation of the receptor tyrosine kinase have been identified as oncogenic drivers in a subset of non-small cell lung cancer (NSCLC). ...

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Ex vivo Culture and Lentiviral Transduction of Benign Prostatic Hyperplasia (BPH) Samples Authors: Urszula Lucja McClurg, Stuart R McCracken, Lisa Butler, Karl T Riabowol and Olivier Binda, date: 11/05/2018, view: 4022, Q&A: 0

To assess oncogenic potential, classical transformation assays are based on cell line models. However, cell line based models do not reflect the complexity of human tissues. We thus developed an inducible expression system for gene expression in ex vivo human tissues, which maintain native tissue architecture, such as epithelia and ...

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Cell Biology

Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types Authors: Sascha Conic, Dominique Desplancq, László Tora and Etienne Weiss, date: 11/05/2018, view: 4436, Q&A: 0

The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous ...

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Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells Authors: Gal Haimovich and Jeffrey E. Gerst, date: 11/05/2018, view: 28416, Q&A: 3

Transcription and RNA decay play critical roles in the process of gene expression and the ability to accurately measure cellular mRNA levels is essential for understanding this regulation. Here, we describe a single-molecule fluorescent in situ hybridization (smFISH) method (as performed in Haimovich et al., 2017) that detects ...

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Staining the Germline in Live Caenorhabditis elegans: Overcoming Challenges by Applying a Fluorescent-dye Feeding Strategy Author: K. Adam Bohnert, date: 11/05/2018, view: 5543, Q&A: 0

C. elegans provides a tractable model organism for studying germline cell biology. Microscopy experiments are relatively facile, as this worm is transparent and germline development can be observed in real-time using DIC microscopy and/or fluorescent transgenes. Despite these many tools, robust staining techniques for imaging germ cells ...

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Immunology

Eicosanoid Isolation from Mouse Intestinal Tissue for ELISA Author: Isabella Rauch, date: 11/05/2018, view: 3504, Q&A: 0

Activation of inflammasomes in peritoneal macrophages and intestinal epithelial cells (IEC) leads to the release of eicosanoids. To assess the amount of eicosanoids released by IEC, lipids need to be isolated from whole tissue previous to analysis by lipid mass spectrometry or ELISA. This protocol describes how to isolate lipids from intestinal ...

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Microbiology

An Innovative Approach to Study Ralstonia solanacearum Pathogenicity in 6 to 7 Days Old Tomato Seedlings by Root Dip Inoculation Authors: Niraj Singh, Rahul Kumar and Suvendra Kumar Ray, date: 11/05/2018, view: 5716, Q&A: 0

Ralstonia solanacearum (F1C1) is a Gram-negative plant pathogenic bacterium that causes lethal wilt disease in a wide range of plant species. This pathogen is very well known for its unpredictable behavior during infection and wilting its host. Because of its mysterious infection behavior, virulence and pathogenicity standardization are ...

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Quantification of Infectious Sendai Virus Using Plaque Assay Authors: Narihito Tatsumoto, Takamasa Miyauchi, Moshe Arditi and Michifumi Yamashita, date: 11/05/2018, view: 5652, Q&A: 0

Sendai virus (SeV) is an enveloped, single-stranded RNA virus of the family Paramyxoviridae. SeV is a useful tool to study its infectious pathomechanism in immunology and the pathomechanism of a murine model of IgA nephropathy. Virus quantification is essential not only to determine the original viral titers for an appropriate ...

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Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-³²P ATP Authors: Heather B. Case and Nicholas E. Dickenson, date: 11/05/2018, view: 3902, Q&A: 0

ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase ...

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Extraction and Analysis of Bacterial Teichoic Acids Authors: Kelvin Kho and Timothy C. Meredith, date: 11/05/2018, view: 7334, Q&A: 0

Teichoic acids (TA) are anionic polymers comprised of polyol phosphate repeat units that are abundant in the cell wall of Gram-positive bacteria. Both wall teichoic acid (WTA) and lipoteichoic acid (LTA) play important roles in regulating cell wall remodeling as well as conferring antibiotic resistance. To analyze TA, we describe a polyacrylamide ...

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Molecular Biology

RNA Immunoprecipitation Assay to Determine the Specificity of SRSF3 Binding to Nanog mRNA Authors: Madara Ratnadiwakara and Minna-Liisa Änko, date: 11/05/2018, view: 6774, Q&A: 0

Mammalian cells express hundreds of RNA binding proteins (RBPs) that are essential regulators of RNA metabolism. RBP activity plays a central role in the control of gene expression programs and identification of RNA-protein interactions is critical for comprehensive understanding of gene regulation in cells. In recent years, various tools and ...

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mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells Authors: Madara Ratnadiwakara and Minna-Liisa Änkö, date: 11/05/2018, view: 26575, Q&A: 2

Gene expression is regulated through multiple steps at both transcriptional and post-transcriptional levels. The net abundance of mature mRNA species in cells is determined by the balance between transcription and degradation. Thus, the regulation of mRNA stability is a key post-transcriptional event that can greatly affect the net level of mRNAs ...

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Generation of Stable Expression Mammalian Cell Lines Using Lentivirus Authors: Neha Tandon, Kaushik N. Thakkar, Edward L LaGory, Yu Liu and Amato J Giaccia, date: 11/05/2018, view: 15394, Q&A: 3

Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3^rd generation lentivirus is very robust and it typically takes about 1-2 ...

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Neuroscience

Eye Drops for Delivery of Bioactive Compounds and BrdU to Stimulate Proliferation and Label Mitotically Active Cells in the Adult Rodent Retina Authors: Cindy L. Linn, Sarah E. Webster and Mark K. Webster, date: 11/05/2018, view: 4286, Q&A: 0

Eye drop treatments are typically used to apply drugs to the anterior structures of the eye. Recently, however, studies have demonstrated that eye drops can reach the retina in the back of the eye if pharmacological agents are carried in appropriate vehicles. Here, we introduce an eye drop procedure to deliver a drug (PNU-282987), in combination ...

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Stem Cell

Generation of BMEC Lines and in vitro BMEC-HSPC Co-culture Assays Authors: Peipei Guo and Shahin Rafii, date: 11/05/2018, view: 5179, Q&A: 0

Endothelial cells (ECs) sustain the self-renewal and regeneration of adult hematopoietic stem and progenitor cells (HSPCs) via deployment of EC-derived paracrine factors, termed as angiocrine factors. Generation of durable ex vivo vascular niche that maintains EC identity and preserves the angiocrine profile of organ of origin offers ...

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Past Issues in 2018

Volume: 8 Issue: 21

Cancer Biology

Cell Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Stem Cell