Volume: 8 Issue: 12 —BIO-PROTOCOL

Biochemistry

Enhancement of Mucus Production in Eukaryotic Cells and Quantification of Adherent Mucus by ELISA Authors: Christian Reuter and Tobias A. Oelschlaeger, date: 06/20/2018, view: 6265, Q&A: 1

The mucosal surfaces of the gastrointestinal, respiratory, reproductive, and urinary tracts, and the surface of the eye harbor a resident microflora that lives in symbiosis with their host and forms a complex ecosystem. The protection of the vulnerable epithelium is primarily achieved by mucins that form a gel-like structure adherent to the apical ...

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Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies Authors: Mayuri Sadoine, Michele Cerminara, Jörg Fitter and Alexandros Katranidis, date: 06/20/2018, view: 3780, Q&A: 0

Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET ...

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Cancer Biology

Isolation of Microvascular Endothelial Cells Authors: Kenneth C.P. Cheung and Federica M. Marelli-Berg, date: 06/20/2018, view: 10020, Q&A: 0

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. Murine endothelial cell culture is an important tool for cardiovascular disease research. This protocol demonstrates a quick, efficient method for the isolation of microvascular endothelial cells from murine ...

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Immunology

Immunohistochemical Identification of Human Skeletal Muscle Macrophages Authors: Kate Kosmac, Bailey D. Peck, R. Grace Walton, Jyothi Mula, Philip A. Kern, Marcas M. Bamman, Richard A. Dennis, Cale A. Jacobs, Christian Lattermann, Darren L. Johnson and Charlotte A. Peterson, date: 06/20/2018, view: 9187, Q&A: 0

Macrophages have well-characterized roles in skeletal muscle repair and regeneration. Relatively little is known regarding the role of resident macrophages in skeletal muscle homeostasis, extracellular matrix remodeling, growth, metabolism and adaptation to various stimuli including exercise and training. Despite speculation into macrophage ...

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Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy Authors: Tan A Nguyen, Lachlan Whitehead and Ken C Pang, date: 06/20/2018, view: 3973, Q&A: 0

Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition ...

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Visualization of RNA at the Single Cell Level by Fluorescent in situ Hybridization Coupled to Flow Cytometry Authors: Alice Coillard and Elodie Segura, date: 06/20/2018, view: 4318, Q&A: 0

The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used ...

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Microbiology

Quantification of the Composition Dynamics of a Maize Root-associated Simplified Bacterial Community and Evaluation of Its Biological Control Effect Authors: Ben Niu and Roberto Kolter, date: 06/20/2018, view: 5133, Q&A: 0

Besides analyzing the composition and dynamics of microbial communities, plant microbiome research aims to understanding the mechanism of plant microbiota assembly and their biological functions. Here, we describe procedures to investigate the role of bacterial interspecies interactions in root microbiome assembly and the beneficial effects of the ...

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A Procedure for Precise Determination of Glutathione Produced by Saccharomyces cerevisiae Authors: Jyumpei Kobayashi, Daisuke Sasaki and Akihiko Kondo, date: 06/20/2018, view: 4733, Q&A: 0

In bioproduction, yields of products must be calculated precisely for accurate evaluation of various fermentation conditions. To evaluate productivity of microorganisms, product amounts per unit of medium volume (e.g., mg-product/L-broth), and/or product amounts per unit of a microorganism amount (e.g., mg-product/mg-dry cell ...

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Characterizing the Transcriptional Effects of Endolysin Treatment on Established Biofilms of Staphylococcus aureus Authors: Lucía Fernández, Silvia González, Diana Gutiérrez, Ana Belén Campelo, Beatriz Martínez, Ana Rodríguez and Pilar García, date: 06/20/2018, view: 3104, Q&A: 0

Biofilms are the most common lifestyle of bacteria in both natural and human environments. The organized structure of these multicellular communities generally protects bacterial cells from external challenges, thereby enhancing their ability to survive treatment with antibiotics or disinfectants. For this reason, the search for new antibiofilm ...

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Implementation of Blue Light Switchable Bacterial Adhesion for Design of Biofilms Authors: Fei Chen and Seraphine V. Wegner, date: 06/20/2018, view: 3797, Q&A: 1

Control of bacterial adhesions to a substrate with high precision in space and time is important to form a well-defined biofilm. Here, we present a method to engineer bacteria such that they adhere specifically to substrates under blue light through the photoswitchable proteins nMag and pMag. This provides exquisite spatiotemporal remote control ...

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ChIP-seq Experiment and Data Analysis in the Cyanobacterium Synechocystis sp. PCC 6803 Authors: Joaquín Giner-Lamia, Miguel A. Hernández-Prieto and Matthias E. Futschik, date: 06/20/2018, view: 7766, Q&A: 0

Nitrogen is an essential nutrient for all living organisms. In cyanobacteria, a group of oxygenic photosynthetic bacteria, nitrogen homeostasis is maintained by an intricate regulatory network around the transcription factor NtcA. Although mechanisms controlling NtcA activity appear to be well understood, the sets of genes under its control (i.e. ...

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Neuroscience

Protocols to Study Declarative Memory Formation in Mice and Humans: Optogenetics and Translational Behavioral Approaches Authors: Azza Sellami, Alice Shaam Al Abed, Laurent Brayda-Bruno, Nicole Etchamendy, Stéphane Valério, Marie Oulé, Laura Pantaléon, Valérie Lamothe, Mylène Potier, Katy Bernard, Maritza Jabourian, Cyril Herry, Nicole Mons and Aline Marighetto, date: 06/20/2018, view: 3768, Q&A: 0

Declarative memory formation depends on the hippocampus and declines in aging. Two functions of the hippocampus, temporal binding and relational organization (Rawlins and Tsaltas, 1983; Eichenbaum et al., 1992; Cohen et al., 1997), are known to decline in aging (Leal and Yassa, 2015). However, in the literature distinct ...

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Buried Food-seeking Test for the Assessment of Olfactory Detection in Mice Authors: Cleiton F. Machado, Thiago M. Reis-Silva, Cassandra S. Lyra, Luciano F. Felicio and Bettina Malnic, date: 06/20/2018, view: 5415, Q&A: 0

The sense of smell allows animals to discriminate a large number of volatile environmental chemicals. Such chemical signaling modulates the behavior of several species that depend on odorant compounds to locate food, recognize territory, predators, and toxic compounds. Olfaction also plays a role in mate choice, mother-infant recognition, and ...

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Plant Science

Analysis of Autophagic Activity Using ATG8 Lipidation Assay in Arabidopsis thaliana Authors: Mengqian Luo and Xiaohong Zhuang, date: 06/20/2018, view: 4793, Q&A: 0

As a fundamental metabolic pathway to degrade and recycle cellular cargos, autophagy is highly induced upon stress, starvation and senescence conditions in plants. A double-membrane structure named autophagosome will form during this process for cargo sequestration and delivery into the vacuole.

A number of regulators have been ...

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Extraction and 16S rRNA Sequence Analysis of Microbiomes Associated with Rice Roots Authors: Joseph Edwards, Christian Santos-Medellín and Venkatesan Sundaresan, date: 06/20/2018, view: 9950, Q&A: 0

Plant roots associate with a wide diversity of bacteria and archaea across the root-soil spectrum. The rhizosphere microbiota, the communities of microbes in the soil adjacent to the root, can contain up to 10 billion bacterial cells per gram of soil (Raynaud and Nunan, 2014) and can play important roles for the fitness of the host plant. Subsets ...

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Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803 Authors: Chiara Gandini, Søren Husted and Sidsel Birkelund Schmidt, date: 06/20/2018, view: 3111, Q&A: 0

Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, ...

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Transient Expression Assay in NahG Arabidopsis Plants Using Agrobacterium tumefaciens Authors: Pepe Cana-Quijada, Eduardo R. Bejarano, Rosa Lozano-Durán and Tábata Rosas-Díaz, date: 06/20/2018, view: 5537, Q&A: 1

Agrobacterium-mediated transient expression has greatly contributed to research in molecular plant biology but has low efficiency and inconsistency in Arabidopsis thaliana (Arabidopsis). Here, we describe a simple, efficient and fast protocol to make transient gene expression in NahG Arabidopsis plants using ...

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Increasing the Membrane Permeability of a Fern with DMSO Author: Marcelo Garcés, date: 06/20/2018, view: 2867, Q&A: 0

Cell membrane prevents the entrance of extra molecules (e.g., transcription and translation inhibitors) into the cell. For studying the physiological effects of transcription and translation inhibitors on Hymenophyllum caudiculatum fronds, we incubate fronds with 0.1% DMSO to test if this increases cell membrane permeability ...

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Stem Cell

Small Molecule-Based Retinal Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Authors: Jie Zhu and Deepak A. Lamba, date: 06/20/2018, view: 4215, Q&A: 0

Retinal degeneration leads to loss of light-sensing photoreceptors eventually resulting in vision impairment and impose a heavy burden on both patients and the society. Currently available treatment options are very limited and mainly palliative. Ever since the discovery of human pluripotent stem cell technologies, cell replacement therapy has ...

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Past Issues in 2018

Volume: 8 Issue: 12

Biochemistry

Cancer Biology

Immunology

Microbiology

Neuroscience

Plant Science

Stem Cell