Mammalia

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    Protocols in Current Issue
    In vitro Infection of Primary Human Monocytes with HIV-1
    Authors:  Patrick O’Connell, Yong-Hui Zheng, Andrea Amalfitano and Yasser A. Aldhamen, date: 07/05/2019, view: 481, Q&A: 0
    [Abstract] Monocyte infection by HIV-1 is an important component of chronic HIV pathogenesis. Following infection by HIV-1, monocytes are able to cross the blood brain barrier and set up a viral reservoir in the central nervous system. Additionally, in the setting of chronic HIV-1 infection, monocytes can become activated either directly through HIV-1 ...
    The Chick Chorioallantoic Membrane (CAM) Assay as a Three-dimensional Model to Study Autophagy in Cancer Cells
    [Abstract] The chick chorioallantoic membrane (CAM) is an extra-embryonic organ and thus well accessible for seeding and harvesting 3D cell cultures. Samples from CAM assays are suitable for protein and gene expression analysis as well as for immuno-histochemical studies. Here we present the CAM assay as a possible model to study autophagy in different types ...
    Glucosidase Inhibition to Study Calnexin-assisted Glycoprotein Folding in Cells
    Authors:  Hao Wang and Qingyu Wu, date: 06/05/2019, view: 477, Q&A: 0
    [Abstract] Calnexin is a chaperone protein that plays a critical role in glycoprotein folding in the endoplasmic reticulum (ER). The function of calnexin depends on its binding to monoglucosylated oligosaccharides on nascent glycoproteins, whereas the generation of monoglucosylated oligosaccharides depends on the activity of α-glucosidases I and II, which ...
    A Flow Cytometric Method to Determine Transfection Efficiency
    [Abstract] Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive ...
    Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR
    Authors:  Bingqian Qu and Stephan Urban, date: 04/05/2019, view: 999, Q&A: 0
    [Abstract] Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and ...
    On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates
    Authors:  Roland Remenyi, Raymond Li and Mark Harris, date: 02/20/2019, view: 1312, Q&A: 0
    [Abstract] Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial ...
    Imaging Higher-order Chromatin Structures in Single Cells Using Stochastic Optical Reconstruction Microscopy
    Authors:  Jianquan Xu and Yang Liu, date: 02/05/2019, view: 1520, Q&A: 0
    [Abstract] Higher-order chromatin organization shaped by epigenetic modifications influence the chromatin environment and subsequently regulate gene expression. Direct visualization of the higher-order chromatin structure at their epigenomic states is of great importance for understanding chromatin compaction and its subsequent effect on gene expression and ...
    Evaluation of Anticancer activity of Silver Nanoparticles on the A549 Human Lung Carcinoma Cell Lines through Alamar Blue Assay
    Authors:  Nikita Sharma, Geeta Arya, R. Mankamna Kumari, Nidhi Gupta and Surendra Nimesh, date: 01/05/2019, view: 2218, Q&A: 0
    [Abstract] Silver nanoparticles have been widely studied to possess antimicrobial as well as anticancer activity, and have found its applications in various fields including pharmaceutical industry, diagnostics, drug delivery, food industry, and others. For this purpose, several cell proliferation assays are widely used for the evaluation of anticancer ...
    Transcytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells
    Authors:  Maria Daniela Garcia-Castillo, Wayne I. Lencer and Daniel J.-F. Chinnapen, date: 10/20/2018, view: 1459, Q&A: 0
    [Abstract] Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific ...
    Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
    Author:  Sébastien Gillotin, date: 10/05/2018, view: 3884, Q&A: 2
    [Abstract] Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes ...



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