Volume: 10 Issue: 16 —BIO-PROTOCOL

Cancer Biology

Measuring Cell Growth and Junction Development in Epithelial Cells Using Electric Cell-Substrate Impedance Sensing (ECIS) Authors: Shaista Anwer and Katalin Szaszi, date: 08/20/2020, view: 2072, Q&A: 0

Electric Cell-substrate Impedance Sensing (ECIS) is an automated method that can be used to quantify processes such as cell attachment, growth, migration and barrier functions (i.e., the properties of tight junctions). The method provides simultaneous information on cell number and tight junction function by detecting electric parameters ...

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Cell Biology

In-vitro GLP-1 Release Assay Using STC-1 Cells Authors: Liu Qi, Tan Shuai, Jia Da, Burstein Ezra and Sifuentes-Dominguez Luis, date: 08/20/2020, view: 1903, Q&A: 0

Enteroendocrine cells (EECs) are known chemosensors in the gastrointestinal (GI) epithelium. They release a diversity of gut hormones in response to various stimuli. Here, we report an in-vitro assay to measure GLP-1 release from cultured murine EEC’s under fatty acid stimulation.

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Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability Authors: Patrizia Gena, Piero Portincasa, Sabino Matera, Yonathan Sonntag, Michael Rutzler and Giuseppe Calamita, date: 08/20/2020, view: 1595, Q&A: 0

Stopped-Flow Light Scattering (SFLS) is a method devised to analyze the kinetics of fast chemical reactions that result in a significant change of the average molecular weight and/or in the shape of the reaction substrates. Several modifications of the original stopped-flow system have been made leading to a significant extension of its technical ...

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Immunology

Neutrophil Extracellular Trap Killing Assay of Candida albicans Authors: Sheng-Yang Wu and Betty A. Wu-Hsieh, date: 08/20/2020, view: 2026, Q&A: 0

Fungal pathogen Candida albicans is one of the top leading causes of overall healthcare-associated bloodstream infections worldwide. Neutrophil is the major effector cell to clear C. albicans infection. Our study showed that mouse neutrophils utilize two independent mechanisms to kill C. albicans: one is CR3 downstream ...

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Real-time in vivo Imaging of LPS-induced Local Inflammation and Drug Deposition in NF-κB Reporter Mice Authors: Artur Schmidtchen and Manoj Puthia, date: 08/20/2020, view: 2143, Q&A: 0

Wound, biomaterial, and surgical infections are all characterized by a localized and excessive inflammation, motivating the development of in vivo methods focused on the analysis of local immune events. However, current inflammation models, such as the commonly used in vivo models of endotoxin-induced inflammation are based on ...

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Microbiology

In vitro Cultivation and Visualization of Malaria Liver Stages in Primary Simian Hepatocytes Authors: Devendra Kumar Gupta and Thierry Diagana, date: 08/20/2020, view: 1619, Q&A: 0

Human liver is the primary and obligatory site for malaria infection where sporozoites invade host hepatocytes. Malaria hepatic stages are asymptomatic and represent an attractive target for development of anti-malarial interventions and vaccines. However, owing to lack of robust and reproducible in vitro culture system, it is difficult ...

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Determination of the Cellular Ion Concentration in Saccharomyces cerevisiae Using ICP-AES Authors: Louise Thines, Anne Iserentant and Pierre Morsomme, date: 08/20/2020, view: 1145, Q&A: 0

The yeast Saccharomyces cerevisiae has been perceived over decades as a highly valuable model organism for the investigation of ion homeostasis. Indeed, many of the genes and biological systems that function in yeast ion homeostasis are conserved throughout unicellular eukaryotes to humans. In this context, measurement of the yeast ...

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Candida albicans Agar Invasion Assays Authors: Shamoon Naseem, Lois M. Douglas and James B. Konopka, date: 08/20/2020, view: 1832, Q&A: 0

The ability of the human fungal pathogen Candida albicans to disseminate into tissues is promoted by a switch from budding to invasive hyphal growth. This morphological transition is stimulated by multiple environmental factors that can vary at different sites of infection. To identify genes that promote invasive growth, C. albicans ...

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Molecular Biology

Mapping mRNA-18S rRNA Contacts Within Translation Initation Complex by Means of Reverse Transcriptase Termination Sites and RNAseq Authors: Irene Díaz-López, René Toribio and Iván Ventoso, date: 08/20/2020, view: 1559, Q&A: 0

The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase ...

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Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues Authors: Siou Ku, Claire Heichette, Laurence Duchesne and Denis Chrétien, date: 08/20/2020, view: 1336, Q&A: 0

Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, ...

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Isolation and Quantification of Extracellular DNA from Biofluids Authors: Ľubica Janovičová, Barbora Konečná, Barbora Vlková and Peter Celec, date: 08/20/2020, view: 1654, Q&A: 0

Extracellular DNA is studied as a diagnostic biomarker, but also as a factor involved in the pathophysiology of several diseases due to its pro-inflammatory properties. Extracellular DNA can be extracted from plasma, urine, saliva or other biofluids using standard DNA isolation procedures and specialized commercial kits. Sample preparation for ...

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Neuroscience

A Reproducible Protocol to Measure the Critical Swimming Speed of Adult Zebrafish Authors: Yuma Wakamatsu, Makoto Kashima and Hiromi Hirata, date: 08/20/2020, view: 1993, Q&A: 1

The quantitative measurement of water flow-induced swimming of fish species using a swimmill is a powerful method to evaluate motor ability of individual fish. Zebrafish is a commonly used vertebrate that enables the study of morphological, physiological and behavioral characteristics associated with genes. We here established a reproducible ...

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Assessing Gα_q/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay Authors: Élie Besserer-Offroy, Rebecca L Brouillette, Jean-Michel Longpré and Philippe Sarret, date: 08/20/2020, view: 1596, Q&A: 0

Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gα_q/15-coupled receptors rely on the use of ...

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Co-culture of Murine Neurons Using a Microfluidic Device for The Study of Tau Misfolding Propagation Authors: Grace I Hallinan, Dianne M Lopez, Mariana Vargas-Caballero, Jonathan West and Katrin Deinhardt, date: 08/20/2020, view: 1690, Q&A: 0

The deposition of misfolded, aggregated tau protein is a hallmark of several neurodegenerative diseases, collectively termed “tauopathies”. Tau pathology spreads throughout the brain along connected pathways in a prion-like manner. The process of tau pathology propagation across circuits is a focus of intense research and has been investigated in ...

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Sequential Reaching Task for the Study of Motor Skills in Monkeys Authors: Machiko Ohbayashi and Nathalie Picard, date: 08/20/2020, view: 1181, Q&A: 0

The ability to perform a sequence of movements is a key component of motor skills, such as typing or playing a musical instrument. How the brain binds elementary movements together into meaningful actions has been a topic of much interest. Here, we describe two sequential reaching tasks that we use to investigate the neural substrate of skilled ...

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Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS Authors: Emma Robinson, James Shorter and Lin Guo, date: 08/20/2020, view: 1825, Q&A: 0

The study of RNA-binding proteins (RBP) offers insight into the mechanisms of pathologic protein aggregation in neurodegenerative diseases. We developed a protocol for purifying an RBP FUS and a nuclear import receptor (NIR) Kapβ2 and testing the ability of Kapβ2 to mitigate FUS aggregation and liquid-liquid phase separation.

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Past Issues in 2020