Protocols in Current Issue
    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: 561, 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: 1095, 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: 1117, 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: 2543, Q&A: 1
    [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 ...
    Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method
    Authors:  Tetsuo Cai and Taisuke Tomita, date: 09/05/2018, view: 1285, Q&A: 0
    [Abstract] Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine ...
    Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation
    Authors:  Eric T. Hall and Stacey K. Ogden, date: 07/05/2018, view: 1479, Q&A: 0
    [Abstract] Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen ...
    Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
    Authors:  Tan A Nguyen, Lachlan Whitehead and Ken C Pang, date: 06/20/2018, view: 1870, Q&A: 0
    [Abstract] Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition ...
    Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining
    [Abstract] In glomerular disease, podocytes and parietal epithelial cells (PECs) are shed in the urine. Therefore, urinary podocytes and PECs are noninvasive biomarkers of glomerular disease. The purpose of this protocol is to employ immunocytochemistry to detect podocytes and PECs, using the WT1 antibody on liquid-based cytology slides.

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