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    Quantification of Neisseria meningitidis Adherence to Human Epithelial Cells by Colony Counting
    [Abstract] To cause an infection, the human specific pathogen Neisseria meningitides must first colonize the nasopharynx. Upon tight interaction with the mucosal epithelium, N. meningitidis may cross the epithelial cellular barrier, reach the bloodstream and cause sepsis and/or meningitis. Since N. meningitidis niche is restricted ...
    Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae
    Authors:  Sviatlana Shashkova, Adam JM Wollman, Stefan Hohmann and Mark C Leake, date: 01/20/2018, view: 413, Q&A: 0
    [Abstract] Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule ...
    High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
    Authors:  Xingmin Aaron Zhang and Thomas F.J. Martin, date: 01/05/2018, view: 291, Q&A: 0
    [Abstract] Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus ...
    Mutant Huntingtin Secretion in Neuro2A Cells and Rat Primary Cortical Neurons
    Authors:  Katarina Trajkovic, Hyunkyung Jeong and Dimitri Krainc, date: 01/05/2018, view: 239, Q&A: 0
    [Abstract] Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary ...
    Using xCELLigence RTCA Instrument to Measure Cell Adhesion
    Authors:  Hellyeh Hamidi, Johanna Lilja and Johanna Ivaska, date: 12/20/2017, view: 962, Q&A: 0
    [Abstract] Cell adhesion to neighbouring cells and to the underlying extracellular matrix (ECM) is a fundamental requirement for the existence of multicellular organisms. As such, the formation, stability and dissociation of cell adhesions are subject to tight control in space and time and perturbations within the sophisticated adhesion machinery are ...
    Generation of Busulfan Chimeric Mice for the Analysis of T Cell Population Dynamics
    Authors:  Thea Hogan, Andrew Yates and Benedict Seddon, date: 12/20/2017, view: 417, Q&A: 0
    [Abstract] This protocol was developed to generate chimeric mice in which T lymphocytes could be stratified by age on the basis of congenic marker expression. The conditioning drug busulfan is used to ablate host haematopoietic stem cells while leaving the peripheral immune system intact. Busulfan treatment is followed by bone marrow transplantation (BMT), ...
    Analysing Temporal Dynamics of T Cell Division in vivo Using Ki67 and BrdU Co-labelling by Flow Cytometry
    Authors:  Thea Hogan, Andrew Yates and Benedict Seddon, date: 12/20/2017, view: 572, Q&A: 0
    [Abstract] This protocol was developed to increase the richness of information available from in vivo T cell proliferation studies. DNA labelling techniques such as BrdU incorporation allow precise control of label administration and withdrawal, so that the division history of a population can be tracked in detail over long timeframes (days-weeks). ...
    Myeloid Progenitor Transformation Assay
    Authors:  Hiroshi Okuda and Akihiko Yokoyama, date: 12/05/2017, view: 512, Q&A: 0
    [Abstract] Numerous oncogenes have been identified to cause leukemia. For example, chromosomal translocation generates various fusion genes of the mixed-lineage leukemia (MLL) gene and a partner gene in leukemia, whose gene products transform primary myeloid progenitors into an immortalized state. To characterize the transforming ability of leukemic ...
    Notch Ligand Binding Assay Using Flow Cytometry
    Authors:  Shweta Varshney and Pamela Stanley, date: 12/05/2017, view: 764, Q&A: 0
    [Abstract] Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most ...
    Quantification of Bacterial Polyhydroxybutyrate Content by Flow Cytometry
    Authors:  Antonio Lagares (Jr.) and Claudio Valverde, date: 12/05/2017, view: 475, Q&A: 0
    [Abstract] We describe here a detailed protocol for the quantification of the intracellular content of polyhydroxybutyrate (PHB) in a population of bacterial cells by flow cytometry, which is based on a consensus of several previously reported works.