Cell Biology


    Protocols in Current Issue
    Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
    Authors:  Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 596, Q&A: 0
    [Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique ...
    In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates
    Authors:  William L. Willis, Abigail Foster, Caitlin Henry, Lai Chu Wu and Wael Jarjour, date: 06/20/2020, view: 488, Q&A: 0
    [Abstract] Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient ...
    Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
    [Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane ...
    Single Cell Volume Measurement Utilizing the Fluorescence Exclusion Method (FXm)
    Authors:  Nash D. Rochman, Kai Yao, Nicolas A. Perez Gonzalez, Denis Wirtz and Sean X. Sun, date: 06/20/2020, view: 641, Q&A: 0
    [Abstract] The measurement of single cell size remains an obstacle towards a deeper understanding of cell growth control, tissue homeostasis, organogenesis, and a wide range of pathologies. Recent advances have placed a spotlight on the importance of cell volume in the regulation of fundamental cell signaling pathways including those known to orchestrate ...
    Live Cell Measurement of the Intracellular pH of Yeast by Flow Cytometry Using a Genetically-Encoded Fluorescent Reporter
    Authors:  Catherine G. Triandafillou and D. Allan Drummond, date: 06/20/2020, view: 524, Q&A: 0
    [Abstract] The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively ...
    Ex vivo Culture Assay Using Human Hair Follicles to Study Circadian Characteristics
    Authors:  Atsuhiro Nishida, Yoshiki Miyawaki, Koichi Node and Makoto Akashi, date: 06/05/2020, view: 494, Q&A: 0
    [Abstract] Ex vivo culture assays of biopsy specimens are advantageous for the experimental evaluation of human circadian characteristics. We developed a simple and non-invasive experimental evaluation method for monitoring the expression of circadian clock genes in an ex vivo culture assay using human hair follicles. This method imposes ...
    Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice
    Authors:  Alissa C. Rothchild, Dat Mai, Alan Aderem and Alan H. Diercks, date: 05/20/2020, view: 928, Q&A: 0
    [Abstract] Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for ...
    Evaluation of B Cell Proliferation in vivo by EdU Incorporation Assay
    Authors:  Adi Biram and Ziv Shulman, date: 05/05/2020, view: 783, Q&A: 0
    [Abstract] Generation of antibodies is crucial for establishing enduring protection from invading pathogens, as well as for maintaining homeostasis with commensal bacteria at mucosal surfaces. Chronic exposure to microbiota- and dietary- derived antigens results in continuous production of antibody producing cells within the Peyer’s patch germinal center ...
    A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase
    Authors:  Ciaran R. McFarlane and James W. Murray, date: 05/05/2020, view: 653, Q&A: 0
    [Abstract] Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay.

    Using the ...
    Assessing in vitro and in vivo Trogocytosis By Murine CD4+ T cells
    Authors:  Jim Reed and Scott A. Wetzel, date: 05/05/2020, view: 740, Q&A: 0
    [Abstract] Recognition of antigens by lymphocytes (B, T, and NK) on the surface of an antigen-presenting cell (APC) leads to lymphocyte activation and the formation of an immunological synapse between the lymphocyte and the APC. At the immunological synapse APC membrane and associated membrane proteins can be transferred to the lymphocyte in a process called ...

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