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Cancer Biology

Invadopodia Detection and Gelatin Degradation Assay Author:  Begoña Díaz, date: 12/20/2013, view: 11104, Q&A: 0
This protocol is designed to quantify invadopodia formation and activity. Invadopodia are protrusive structures elaborated by cancer cells that mediate cell attachment and remodeling of the extracellular matrix. These structures contribute to the ability of cancer cells to invade and metastasize. In this protocol, both the presence of invadopodia ...
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Measurement of Acetylcholine from Cell Lines Authors:  Jamie K. Lau, Kathleen C. Brown and Piyali Dasgupta, date: 12/20/2013, view: 5241, Q&A: 0
Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80-90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in ...
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Cell Biology

ImmunoFISH for Adherent Cultured Mammalian Cells Authors:  Francesca Rossiello, Marzia Fumagalli and Fabrizio d’Adda di Fagagna, date: 12/20/2013, view: 5215, Q&A: 0
This protocol is optimized for immunoFISH staining of adherent cultured mammalian cells. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) and FISH against telomeric DNA.
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ImmunoFISH for Mice and Baboons Frozen Sections Authors:  Francesca Rossiello, Marzia Fumagalli and Fabrizio d’Adda di Fagagna, date: 12/20/2013, view: 4289, Q&A: 0
This protocol is optimized for immunoFISH staining of OCT section of mouse tissues. It combines immunofluorescence for DNA damage response factors (e.g. 53BP1) (Le et al., 2010) and FISH against telomeric DNA.
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Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay) Authors:  Xin Zhang and Qingsong Liu, date: 12/20/2013, view: 13612, Q&A: 0
Autophagy is a dynamic cellular event that is involved in the degradation of long lived proteins and organelles in cells. Biochemical methods such as western blot to measure autophagic proteins have been increasingly used in autophagy studies because it is convenient and objective. Among them, the total amount of Microtubule-associated protein ...
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Immunology

Influenza Virus-cell Fusion Inhibition Assay Authors:  Mayo Yasugi and Kazuyoshi Ikuta, date: 12/20/2013, view: 5134, Q&A: 0
During viral infection to host cells, several viruses undergo the process of endocytosis and pH-dependent fusion. By fusion of viral membrane with host cellular membrane, the viral core invades to host cytoplasm. A part of monoclonal antibodies against viral membrane protein have potential to inhibit the viral fusion step. Here we describe in ...
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Isolation of Neutralizing Antibody Authors:  Mayo Yasugi and Kazuyoshi Ikuta, date: 12/20/2013, view: 4297, Q&A: 0
Use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of specific pathogens and their antigenicity. Especially, Human MAbs (HuMAbs) with neutralizing activity against specific virus could have potential therapeutic application, and provide significant information on human epitopes that could be important ...
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Surface Plasmon Resonance Analysis of Antigen-Antibody Interaction Authors:  Mitsuhiro Nishimura and Kazuyoshi Ikuta, date: 12/20/2013, view: 6905, Q&A: 0
Molecular interaction between monoclonal antibodies (MAbs) and their recognized antigen is a fundamental event leading to the neutralization activity. Estimation of their binding affinity gives beneficial information to characterize the MAbs and to develop more effective MAbs. Surface plasmon resonance (SPR) analysis is a powerful tool to analyze ...
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Microbiology

EMS Mutagenesis of Clostridium difficile to Identify Strains with Germination-null Phenotypes Authors:  Michael B. Francis and Joseph A. Sorg, date: 12/20/2013, view: 5021, Q&A: 0
Clostridium difficile is a Gram-positive, spore-forming, strict anaerobe and the leading cause of antibiotic-associated diarrhea (McFarland, 2008). Germination by C. difficile spores is the first step in pathogenesis. Thus, identifying mechanisms of C. difficile spore germination may lead to novel anti-germination ...
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Virulence Studies of Clostridium difficile Authors:  Michael B. Francis and Joseph A. Sorg, date: 12/20/2013, view: 4400, Q&A: 0
Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) can colonize antibiotic-treated hosts (McFarland, 2008). Antibiotics alter the composition of the normal, benign microbial flora which leads to loss of colonization resistance (Wilson and Perini, 1988; Antonopoulos et al., 2009). C. difficile spores ...
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Colony Forming Assay for HCV-Replicon Cell Line Authors:  Chang Ho Lee and Seong-Wook Lee, date: 12/20/2013, view: 4821, Q&A: 0
Hepatitis C virus (HCV) is the main causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Since the HCV genome is present exclusively in RNA form during replication, a number of anti-HCV drugs show appearance of rapid drug-resistant viruses. Therefore, it is important to test generation of drug-escape mutant viruses ...
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Neuroscience

Ex utero Electroporation into Mouse Embryonic Neocortex Author:  Branden R. Nelson, date: 12/20/2013, view: 5899, Q&A: 0
This technique allows highly efficient and reproducible transfer of DNA/RNA into the embryonic neocortex of rodents across multiple ages. Ex utero electroporation compliments the more technically difficult in utero electroporation technique by maximizing the number of embryos for available for a given experiment, as well as ...
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Plant Science

Observation of Chloroplast-actin Filaments in Leaves of Arabidopsis Authors:  Sam-Geun Kong and Masamitsu Wada , date: 12/20/2013, view: 4859, Q&A: 0
Chloroplast-actin (cp-actin) filaments play a pivotal role in chloroplast photorelocation movement. This protocol describes observation of cp-actin filaments in intact palisade cells of Arabidopsis leaves (Kong et al., 2013). The live cell imaging of cp-actin filaments is taken on moving chloroplasts, so that this protocol is ...
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Library Construction for Genome-wide Bisulfite Sequencing in Plants Authors:  David Moraga Amador, Chenggang Wang, Kevin H. Holland and Zhonglin Mou, date: 12/20/2013, view: 7517, Q&A: 0
DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. ...
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Membrane Preparation, Sucrose Density Gradients and Two-phase Separation Fractionation from Five-day-old Arabidopsis seedlings Authors:  Haibing Yang and Angus Murphy, date: 12/20/2013, view: 11874, Q&A: 0
Membrane preparation has been widely used for characterization the membrane proteins. Membrane fractions can be separated by a combination of differential and density-gradient centrifugation techniques (Hodges et al., 1972; Leonard and Vanderwoude, 1976). Here we firstly describe a method to isolate total microsomal fractions including ...
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Stem Cell

Preparation of Adult Mouse Muscle Stem Cells Author:  Ju Li, date: 12/20/2013, view: 5581, Q&A: 0
Muscle stem cells are adult stem cells responsible for muscle development, growth and regeneration. Current knowledge suggests those cells are heterogeneous population shared their position between the sarcolemma and basal lamina of muscle fibers. This protocol describes the technique to dissociate and collect the stem cells from skeletal muscle ...
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Intestinal Differentiation of Human ESCs Authors:  Soichiro Ogaki and Shoen Kume, date: 12/20/2013, view: 3818, Q&A: 0
ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of ...
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Intestinal Differentiation of Mouse ESCs Authors:  Soichiro Ogaki and Shoen Kume, date: 12/20/2013, view: 3737, Q&A: 0
ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of ...
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