Mammalia

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    Protocols in Current Issue
    A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery
    Authors:  Matthew J. Stopford, Scott P. Allen and Laura Ferraiuolo, date: 09/05/2019, view: 396, Q&A: 0
    Amyotrophic lateral sclerosis (ALS) is an adult onset neurological disorder characterized by loss of motor neurons leading to progressive muscle wasting and eventually death. Astrocytes play a key role in disease pathogenesis. However, the ability ...
    A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material
    We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from ...
    In vitro Intestinal Epithelial Wound-healing Assays Using Electric Cell-Substrate Impedance Sensing Instrument
    Authors:  Olivier Merlin-Zhang, Junsik Sung and Emilie Viennois, date: 09/05/2019, view: 206, Q&A: 0
    Here, we describe an in vitro epithelial wound-healing assay using Electric Cell-Substrate Impedance Sensing (ECIS) technology. The ECIS technology is a real time cell growth assay based on a small (250 μm diameter) active gold electrode ...
    Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells
    Authors:  Stephanie Dooves, Aishwarya G. Nadadhur, Lisa Gasparotto and Vivi M. Heine, date: 09/05/2019, view: 295, Q&A: 0
    Crosstalk between neurons and oligodendrocytes is important for proper brain functioning. Multiple co-culture methods have been developed to study oligodendrocyte maturation, myelination or the effect of oligodendrocytes on neurons. However, most of ...
    A Robust, One-step FRET Assay for Human Heparanase
    Authors:  Jyothi C. Sistla and Umesh R. Desai, date: 09/05/2019, view: 170, Q&A: 0
    Heparanase, an endo-β-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains at distinct sites and plays important biological roles including modulation of cell growth and metastasis. Although a number of ...
    Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides
    Authors:  Masatoshi Murai and Hideto Miyoshi, date: 09/05/2019, view: 135, Q&A: 0
    The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the ...
    Isolation of Neutrophil Nuclei for Use in NETosis Assays
    Author:  Dave Boucher, date: 09/05/2019, view: 269, Q&A: 0
    Neutrophils are critical immune cells that protect our body against invading pathogens. They generate antibacterial DNA structures called neutrophil extracellular traps (NET). Recently we identified a new mechanism that enables NET formation. We ...
    Estrous Cycle Monitoring in Mice with Rapid Data Visualization and Analysis
    Authors:  Leanna K. Pantier, Jiang Li and Catherine A. Christian, date: 09/05/2019, view: 318, Q&A: 0
    The estrous cycle provides a readout of reproductive health in female laboratory rodents, and estrous cycle stage can be an important physiological variable. Accurate assessment of estrous cycle stage is also important in producing timed pregnancies ...
    A Novel Dual Lentiviral CRISPR-based Transcriptional Activation System for Gene Expression Regulation in Neurons
    Authors:  Katherine E. Savell, Faraz A. Sultan and Jeremy J. Day, date: 09/05/2019, view: 523, Q&A: 0
    Robust and efficient gene expression control enables the study of a gene’s function in the central nervous system. Advances in CRISPR-based technology provide new avenues not only for gene editing, but for complex transcriptional control. Here, we ...
    A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery
    Authors:  Matthew J. Stopford, Scott P. Allen and Laura Ferraiuolo, date: 09/05/2019, view: 396, Q&A: 0
    [Abstract] Amyotrophic lateral sclerosis (ALS) is an adult onset neurological disorder characterized by loss of motor neurons leading to progressive muscle wasting and eventually death. Astrocytes play a key role in disease pathogenesis. However, the ability to study astrocytic support towards motor neurons in ALS has been limited by a lack of sustainable ...
    A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material
    [Abstract] We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin ...
    In vitro Intestinal Epithelial Wound-healing Assays Using Electric Cell-Substrate Impedance Sensing Instrument
    Authors:  Olivier Merlin-Zhang, Junsik Sung and Emilie Viennois, date: 09/05/2019, view: 206, Q&A: 0
    [Abstract] Here, we describe an in vitro epithelial wound-healing assay using Electric Cell-Substrate Impedance Sensing (ECIS) technology. The ECIS technology is a real time cell growth assay based on a small (250 μm diameter) active gold electrode which resistance is measured continuously. When intestinal epithelial cells reach confluency on the ...
    Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells
    Authors:  Stephanie Dooves, Aishwarya G. Nadadhur, Lisa Gasparotto and Vivi M. Heine, date: 09/05/2019, view: 295, Q&A: 0
    [Abstract] Crosstalk between neurons and oligodendrocytes is important for proper brain functioning. Multiple co-culture methods have been developed to study oligodendrocyte maturation, myelination or the effect of oligodendrocytes on neurons. However, most of these methods contain cells derived from animal models. In the current protocol, we co-culture ...
    A Robust, One-step FRET Assay for Human Heparanase
    Authors:  Jyothi C. Sistla and Umesh R. Desai, date: 09/05/2019, view: 170, Q&A: 0
    [Abstract] Heparanase, an endo-β-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains at distinct sites and plays important biological roles including modulation of cell growth and metastasis. Although a number of different types of heparanase assays have been reported to date, most are labor intensive, complex ...
    Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides
    Authors:  Masatoshi Murai and Hideto Miyoshi, date: 09/05/2019, view: 135, Q&A: 0
    [Abstract] The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [125 ...
    Isolation of Neutrophil Nuclei for Use in NETosis Assays
    Author:  Dave Boucher, date: 09/05/2019, view: 269, Q&A: 0
    [Abstract] Neutrophils are critical immune cells that protect our body against invading pathogens. They generate antibacterial DNA structures called neutrophil extracellular traps (NET). Recently we identified a new mechanism that enables NET formation. We observed that following recognition of lipopolysaccharides, inflammatory caspases cleave Gasdermin D ...
    Estrous Cycle Monitoring in Mice with Rapid Data Visualization and Analysis
    Authors:  Leanna K. Pantier, Jiang Li and Catherine A. Christian, date: 09/05/2019, view: 318, Q&A: 0
    [Abstract] The estrous cycle provides a readout of reproductive health in female laboratory rodents, and estrous cycle stage can be an important physiological variable. Accurate assessment of estrous cycle stage is also important in producing timed pregnancies for developmental studies. Here, we provide a protocol for evaluation of estrous cycle stage ...
    A Novel Dual Lentiviral CRISPR-based Transcriptional Activation System for Gene Expression Regulation in Neurons
    Authors:  Katherine E. Savell, Faraz A. Sultan and Jeremy J. Day, date: 09/05/2019, view: 523, Q&A: 0
    [Abstract] Robust and efficient gene expression control enables the study of a gene’s function in the central nervous system. Advances in CRISPR-based technology provide new avenues not only for gene editing, but for complex transcriptional control. Here, we describe a protocol to generate high-titer lentiviruses with neuron-optimized CRISPR-activation ...
    Application of a Modified Smart-seq2 Sample Preparation Protocol for Rare Cell Full-length Single-cell mRNA Sequencing to Mouse Oocytes
    Authors:  Rebecca S. Treger, Scott D. Pope, Xiaojun Xing and Akiko Iwasaki, date: 08/20/2019, view: 258, Q&A: 0
    [Abstract] Endogenous retroviruses (ERV) are transposable retroelements that form ~10% of the murine genome and whose family members are differentially expressed throughout embryogenesis. However, precise regulation of ERV in germ cells remains unclear. To investigate ERV expression in oocytes, we adapted a single-cell mRNA-sequencing library preparation ...



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