Mammalia

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    Protocols in Current Issue
    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: 40, 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: 302, 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: 534, 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: 592, 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: 1121, Q&A: 0
    [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 ...
    Qualitative in vivo Bioluminescence Imaging
    Authors:  Devbarna Sinha, Zalitha Pieterse and Pritinder Kaur, date: 09/20/2018, view: 1032, Q&A: 0
    [Abstract] Bioluminescence imaging (BLI) technology is an advanced method of carrying out molecular imaging on live laboratory animals in vivo. This powerful technique is widely-used in studying a variety of biological processes, and it has been an ideal tool in exploring tumor growth and metastatic spread in real-time. This technique ensures the ...
    Cell Synchronization by Double Thymidine Block
    Authors:  Guo Chen and Xingming Deng, date: 09/05/2018, view: 4945, Q&A: 7
    [Abstract] Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at ...
    Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells
    Authors:  Nicolas Lemus-Diaz, Liezel Tamon and Jens Gruber, date: 09/05/2018, view: 1863, Q&A: 0
    [Abstract] MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with ...
    Hypoxia Reporter Element Assay
    Authors:  Daelynn R Buelow and Sharyn D. D Baker, date: 08/05/2018, view: 872, Q&A: 0
    [Abstract] Hypoxia is a condition in which there is a decrease in oxygen supply to the cellular environment. Changes to cellular oxygen levels can lead to transcriptional changes of oxygen-regulated genes. Reporter assays are used to study gene expression alteration and modifications in response to environmental changes. Dual-reporter assays allow the ...
    Fluorophore-Based Mitochondrial Ca2+ Uptake Assay
    Author:  Charles B. Phillips, date: 07/20/2018, view: 943, Q&A: 0
    [Abstract] The physiological importance of mitochondrial calcium uptake, observed in processes such as ATP production, intracellular calcium signaling, and apoptosis, makes desirable a simple, straightforward way of investigating this event with unambiguous results. The following protocol uses a calcium-sensitive, membrane-impermeable fluorophore to monitor ...



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