Protocols in Current Issue
    Isolation and Transcriptomic Profiling of Single Myofibers from Mice
    [Abstract] Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of ...
    Quantitation of TLR4 Internalization in Response to LPS in Thioglycollate Elicited Peritoneal Mouse Macrophages by Flow Cytometry
    Authors:  Katharina Richard, Stefanie N. Vogel and Darren J. Perkins, date: 09/20/2019, view: 231, Q&A: 0
    [Abstract] Internalization of cell surface Toll-like Receptor 4 (TLR4) is a convenient read-out to measure LPS dependent activation of the TRIF adaptor pathway. We here provide a protocol to quantify the LPS dependent internalization of TLR4 using thioglycollate-elicited peritoneal macrophages by flow cytometry.
    Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells
    Authors:  Xueer Jiang, Ingrid Brust-Mascher and Li-En Jao, date: 08/20/2019, view: 668, Q&A: 0
    [Abstract] Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of ...
    Production and Purification of Dengue Virus-like Particles from COS-1 Cells
    Authors:  Jedhan Ucat Galula, Gwong-Jen J. Chang and Day-Yu Chao, date: 06/20/2019, view: 941, Q&A: 0
    [Abstract] Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. ...
    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 ...
    Primary Embryonic Rat Cortical Neuronal Culture and Chronic Rotenone Treatment in Microfluidic Culture Devices
    Authors:  Victor S. Van Laar, Beth Arnold and Sarah B Berman, date: 03/20/2019, view: 1297, Q&A: 0
    [Abstract] In the study of neurodegenerative diseases, it is imperative to study the cellular and molecular changes associated with pathogenesis in the relevant cell type, central nervous system neurons. The unique compartmentalized morphology and bioenergetic needs of primary neurons present complications for their study in culture. Recent microculture ...
    DNA Immunization Using in vivo Electroporation for Generating Monoclonal Antibodies Against Mouse IL-9R
    [Abstract] Membrane proteins such as cytokine receptors and G protein-coupled receptors can be drug targets. Recently, we have generated specific monoclonal antibodies (mAbs) against the mouse IL-9 receptor (IL-9R) and found that IL-9R on memory B cells have critical roles in T-dependent immune response. So far, most antibodies against cell surface proteins ...
    Induced Germinal Center B Cell Culture System
    Authors:  Kei Haniuda and Daisuke Kitamura, date: 02/20/2019, view: 2622, Q&A: 0
    [Abstract] The germinal center (GC) is the site where B cells undergo clonal expansion, affinity-based selection, and differentiation into memory B cells or plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells in vivo ...
    Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types
    Authors:  Sascha Conic, Dominique Desplancq, László Tora and Etienne Weiss, date: 11/05/2018, view: 1693, Q&A: 0
    [Abstract] The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous ...
    Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
    Author:  Sébastien Gillotin, date: 10/05/2018, view: 5019, 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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