Mammalia

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    Protocols in Current Issue
    In vitro Measurement of Membrane Attack Complex in RPE Cells
    Authors:  Kelly Mulfaul and Sarah L. Doyle, date: 02/20/2021, view: 533, Q&A: 0

    Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a

    ...
    RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities
    Authors:  Juan Pablo Tosar, Fabiana Gámbaro, Mauricio Castellano and Alfonso Cayota, date: 02/20/2021, view: 673, Q&A: 0

    Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and

    ...
    Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix
    Authors:  Zihan Ling, Yunhui Xing, Emily Reinoso Jacome, Shierly W. Fok and Xi Ren, date: 02/20/2021, view: 538, Q&A: 0

    Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the

    ...
    Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry
    Authors:  Rong Xu, Yekai Wang and Jianhai Du, date: 02/20/2021, view: 571, Q&A: 0

    Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis

    ...
    An Imaging Flow Cytometry Method to Measure Citrullination of H4 Histone as a Read-out for Neutrophil Extracellular Traps Formation
    Authors:  Emilia A. Barbu, Venina M. Dominical, Laurel Mendelsohn and Swee Lay Thein, date: 02/20/2021, view: 529, Q&A: 0

    The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation

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    Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
    Authors:  Ryota Futamata, Noriyuki Kioka and Kazumitsu Ueda, date: 02/20/2021, view: 672, Q&A: 0

    The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured

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    Live Cell FRET Analysis of the Conformational Changes of Human P-glycoprotein
    Authors:  Ryota Futamata, Noriyuki Kioka and Kazumitsu Ueda, date: 02/20/2021, view: 672, Q&A: 0
    [Abstract]

    The molecular mechanisms of P-glycoprotein (P-gp; also known as MDR1 or ABCB1) have been mainly investigated using artificial membranes such as lipid-detergent mixed micelles, artificial lipid bilayers, and membrane vesicles derived from cultured cells. Although these in vitro experiments help illustrate details about the molecular mechanisms of

    ...
    An Imaging Flow Cytometry Method to Measure Citrullination of H4 Histone as a Read-out for Neutrophil Extracellular Traps Formation
    Authors:  Emilia A. Barbu, Venina M. Dominical, Laurel Mendelsohn and Swee Lay Thein, date: 02/20/2021, view: 529, Q&A: 0
    [Abstract]

    The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy

    ...
    Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry
    Authors:  Rong Xu, Yekai Wang and Jianhai Du, date: 02/20/2021, view: 571, Q&A: 0
    [Abstract]

    Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis of amino acids and nucleotides. Both nuclear magnetic resonance spectroscopy and mass spectrometry

    ...
    Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix
    Authors:  Zihan Ling, Yunhui Xing, Emily Reinoso Jacome, Shierly W. Fok and Xi Ren, date: 02/20/2021, view: 538, Q&A: 0
    [Abstract]

    Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the immobilization of bioactive molecules. To enable ECM functionalization in a chemoselective manner, we have

    ...
    RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities
    Authors:  Juan Pablo Tosar, Fabiana Gámbaro, Mauricio Castellano and Alfonso Cayota, date: 02/20/2021, view: 673, Q&A: 0
    [Abstract]

    Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and remain in ultracentrifugation supernatants of cell-conditioned medium or blood serum. We have observed

    ...
    In vitro Measurement of Membrane Attack Complex in RPE Cells
    Authors:  Kelly Mulfaul and Sarah L. Doyle, date: 02/20/2021, view: 533, Q&A: 0
    [Abstract]

    Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the

    ...
    Rapid and Simplified Induction of Neural Stem/Progenitor Cells (NSCs/NPCs) and Neurons from Human Induced Pluripotent Stem Cells (hiPSCs)
    Authors:  Ryutaro Kajihara, Tadahiro Numakawa and Takumi Era, date: 02/05/2021, view: 232, Q&A: 0
    [Abstract]

    Human induced pluripotent stem cells (iPSCs) and their progeny displaying tissue-specific characteristics have paved the way for regenerative medicine and research in various fields such as the elucidation of the pathological mechanism of diseases and the discovery of drug candidates. iPSC-derived neurons are particularly valuable as it is

    ...
    Relative Quantification of NaV1.1 Protein in Mouse Brains Using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) Method
    Authors:  Zhou Han, Anne Christiansen, Meena and Gene Liau, date: 02/05/2021, view: 187, Q&A: 0
    [Abstract]

    Densitometric analysis is often used to quantify NaV1.1 protein on immunoblots, although the sensitivity and dilution linearity of the method are usually poor. Here we present a protocol for quantification of NaV1.1 in mouse brain tissues using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL is based on ELISA

    ...
    Isolation of Murine Primary Aortic Smooth Muscle Cells
    Authors:  Max Ole Hubert, Juan Rodriguez-Vita, Lena Wiedmann and Andreas Fischer, date: 02/05/2021, view: 218, Q&A: 0
    [Abstract]

    Vascular smooth muscle cells (VSMCs) have been cultured for decades to study the role of these cells in cardiovascular disorders. The most common source of VSMCs is the rat aorta. Here we show the adaptation of this method to isolate and culture mouse aortic VSMCs. The advantage of this method is that there are many more transgenic mouse lines

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    Induction of Epithelial-mesenchymal Transition in MDCK II Cells
    Authors:  Agnieszka Pastuła and Richard Lundmark, date: 02/05/2021, view: 266, Q&A: 0
    [Abstract]

    Epithelial-mesenchymal transition (EMT) is a reversible process of epithelial cell transdifferentiation into a mesenchymal cell, that enables initiation of cell migration. EMT plays an important role in embryonic development, tissue repair and cancer metastasis. Better understanding of cellular and molecular events during EMT will not only provide

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