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    Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor
    [Abstract] Reactive oxygen species (ROS) are not only known for their toxic effects on cells, but they also play an important role as second messengers. As such, they control a variety of cellular functions such as proliferation, metabolism, differentiation and apoptosis. Thus, ROS are involved in the regulation of multiple physiological and ...
    Analysis of Exosome Transfer in Mammalian Cells by Fluorescence Recovery after Photobleaching
    [Abstract] During the course of evolution, prokaryote and eukaryote cells have developed elegant and to some extent analogous strategies to communicate with each other and to adapt to their surrounding environment. Eukaryotic cells communicate with each other through direct interaction via juxtracrine signaling and/or by secreting soluble factors. These ...
    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: 175, 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 ...
    The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
    Authors:  Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 169, Q&A: 0
    [Abstract] While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding ...
    Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions
    Authors:  Yohei Katoh, Kentaro Nakamura and Kazuhisa Nakayama, date: 01/05/2018, view: 281, Q&A: 0
    [Abstract] The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can ...
    A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 12/20/2017, view: 510, Q&A: 0
    [Abstract] In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and ...
    Using xCELLigence RTCA Instrument to Measure Cell Adhesion
    Authors:  Hellyeh Hamidi, Johanna Lilja and Johanna Ivaska, date: 12/20/2017, view: 736, 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 ...
    Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells
    Authors:  Florent Ubelmann, Tatiana Burrinha and Claudia Guimas Almeida, date: 12/05/2017, view: 427, Q&A: 0
    [Abstract] The established primary trigger of Alzheimer’s disease’s is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane (Koo et al., 1996; Ubelmann et ...
    GC/MS-based Analysis of Volatile Metabolic Profile Along in vitro Differentiation of Human Induced Pluripotent Stem Cells
    [Abstract] Human induced pluripotent stem cells (hiPSCs) are a promising tool in cell-based therapies for degenerative diseases. A safe application of hiPSCs in vivo, requires the detection of the presence of residual undifferentiated pluripotent cells that can potentially cause the insurgence of teratomas. Several studies point out that metabolic ...
    Cell-free Generation of COPII-coated Procollagen I Carriers
    Authors:  Lin Yuan, Satoshi Baba, Kanika Bajaj and Randy Schekman, date: 11/20/2017, view: 581, Q&A: 0
    [Abstract] The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the ...