Volume: 10 Issue: 3 —BIO-PROTOCOL

Biophysics

Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces Authors: Roberta M Sabino and Ketul C Popat, date: 02/05/2020, view: 2150, Q&A: 0

Biomaterial-associated thrombosis is still a major concern for blood-contacting implants. After the medical device is implanted and comes in contact with blood, several complex reactions occur, which may lead to thrombus formation and failure of the device. Therefore, it is essential to evaluate the biomaterial interaction with the whole blood. ...

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Cancer Biology

Approaching RNA-seq for Cell Line Identification Authors: Tabrez A. Mohammad and Yidong Chen, date: 02/05/2020, view: 1746, Q&A: 0

Cancer cell lines serve as invaluable model systems for cancer biology research and help in evaluating the efficacy of new therapeutic agents. However, cell line contamination and misidentification have become one of the most pressing problems affecting biomedical research. Available methods of cell line authentication suffer from limited access, ...

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Detection of Individual RNA in Fixed Cells and Tissues by Chromogenic ISH Authors: Meng Jiang, Chen Lin and Rongqin Ke, date: 02/05/2020, view: 1640, Q&A: 0

Visualization of RNA molecules in situ helps to better understand the functions of expressed genes. Currently, most conventional in situ hybridization methods for visualization of individual RNAs are based on fluorescence detection. Herein we present a chromogenic in situ hybridization protocol for visualization of ...

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Measurement of ATPase Activity of Valosin-containing Protein/p97 Authors: Kruthi Suvarna, Kaori Honda, Makoto Muroi, Yasumitsu Kondoh, Hiroyuki Osada and Nobumoto Watanabe, date: 02/05/2020, view: 1931, Q&A: 0

Valosin-containing protein (VCP; also known as p97) is a type II ATPase regulating several cellular processes. Using proteomic techniques, we identified a chemical compound that binds to the D1 ATPase domain of VCP. The protocol described here was to study the effect of the compound on ATPase activity in vitro of purified VCP protein. ...

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Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry Authors: Kruthi Suvarna, Kaori Honda, Makoto Muroi, Yasumitsu Kondoh, Nobumoto Watanabe and Hiroyuki Osada, date: 02/05/2020, view: 1915, Q&A: 0

Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several ...

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Cell Biology

Live Mitochondrial or Cytosolic Calcium Imaging Using Genetically-encoded Cameleon Indicator in Mammalian Cells Authors: Elisa Greotti and Tullio Pozzan, date: 02/05/2020, view: 2473, Q&A: 0

Calcium (Ca²⁺) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca²⁺]) in different pathophysiological conditions. Ca²⁺ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex ...

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Rapid Nickel-based Isolation of Extracellular Vesicles from Different Biological Fluids Authors: Michela Notarangelo, Deborah Ferrara, Cristina Potrich, Lorenzo Lunelli, Lia Vanzetti, Alessandro Provenzani, Manuela Basso, Alessandro Quattrone and Vito G. D'Agostino, date: 02/05/2020, view: 2197, Q&A: 0

Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a ...

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Developmental Biology

Mouse Embryonic Tooth Germ Dissection and Ex vivo Culture Protocol Authors: Xue Han, Keigo Yoshizaki, Tian Tian, Kanako Miyazaki, Ichiro Takahashi and Satoshi Fukumoto, date: 02/05/2020, view: 2065, Q&A: 1

A tooth germ ex vivo organ culture allows visualization of its development in different stages, thus enabling investigation of the molecular mechanisms of regulatory factors. Tooth germs can be rapidly dissected from E13 mouse embryos and placed on cell culture inserts for observation of subsequent tooth germ development in a ...

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Microbiology

Transcervical Mouse Infections with Chlamydia trachomatis and Determination of Bacterial Burden Authors: Karthika Rajeeve and Rajeeve Sivadasan, date: 02/05/2020, view: 1944, Q&A: 0

Chlamydia trachomatis is an obligate human pathogen. It infects the genital tract of humans ascending into the fallopian tube, exacerbated by chronic pelvic pain, pelvic inflammatory disease, and fallopian tube scaring resulting in infertility and other malignancies. The major hurdle in controlling chlamydial spread is that the ...

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Rapid Detection of Proliferative Bacteria by Electrical Stimulation Authors: Conor LA Edwards, Dmitry Malyshev, James P Stratford and Munehiro Asally, date: 02/05/2020, view: 2409, Q&A: 0

Detecting live bacteria is an important task for antimicrobial susceptibility testing (AST) in the medical sector and for quality-monitoring in biological industries. Current methods for live-bacteria detection suffer limitations in speed or sensitivity. In a recent paper, we reported that electrical response dynamics in membrane potential enable ...

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Expression and Purification of Adeno-associated Virus Virus-like Particles in a Baculovirus System and AAVR Ectodomain Constructs in E. coli Authors: Nancy Meyer, Omar Davulcu, Qing Xie, Mark Silveria, Grant M. Zane, Edward Large and Michael S. Chapman, date: 02/05/2020, view: 2203, Q&A: 0

Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 ...

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Competition Assays to Quantify the Effect of Biocontrol Yeasts against Plant Pathogenic Fungi on Fruits Authors: Electine Magoye, Melanie Pfister, Maja Hilber-Bodmer and Florian M. Freimoser, date: 02/05/2020, view: 1441, Q&A: 0

Yeasts such as Aureobasidium pullulans are unicellular fungi that occur in all environments and play important roles in biotechnology, medicine, food and beverage production, research, and agriculture. In the latter, yeasts are explored as biocontrol agents for the control of plant pathogenic fungi (e.g., Botrytis cinerea, ...

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Molecular Biology

Structural Alignment and Covariation Analysis of RNA Sequences Authors: Nicolas J. Tourasse and Fabien Darfeuille, date: 02/05/2020, view: 2049, Q&A: 0

RNA molecules adopt defined structural conformations that are essential to exert their function. During the course of evolution, the structure of a given RNA can be maintained via compensatory base-pair changes that occur among covarying nucleotides in paired regions. Therefore, for comparative, structural, and evolutionary studies of RNA ...

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Neuroscience

Implanting and Recycling Neuropixels Probes for Recordings in Freely Moving Mice Authors: Ashley L. Juavinett, George Bekheet and Anne K. Churchland, date: 02/05/2020, view: 2774, Q&A: 0

Recording neural activity in unrestricted animals is necessary to unravel the neural basis of ethological behaviors. Recently, Neuropixels probes have made important strides in improving yield and lowering noise, but have limited use cases in freely moving animals. Although there are a number of studies demonstrating the use of these probes in ...

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Stem Cell

In vitro Self-organized Mouse Small Intestinal Epithelial Monolayer Protocol Authors: Gizem Altay, Eduard Batlle, Vanesa Fernández-Majada and Elena Martinez, date: 02/05/2020, view: 4012, Q&A: 0

Developing protocols to obtain intestinal epithelial monolayers that recapitulate in vivo physiology to overcome the limitations of the organoids’ closed geometry has become of great interest during the last few years. Most of the developed culture models showed physiological-relevant cell composition but did not prove self-renewing ...

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Past Issues in 2020

Volume: 10 Issue: 3

Biophysics

Cancer Biology

Cell Biology

Developmental Biology

Microbiology

Molecular Biology

Neuroscience

Stem Cell