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
    [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 ...
    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: 522, 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 ...
    In vitro Differentiation of Thymic Treg Cell Progenitors to Mature Thymic Treg Cells
    Authors:  David L. Owen and Michael A. Farrar, date: 08/20/2019, view: 218, Q&A: 0
    [Abstract] Thymic Treg cell differentiation occurs via a two-step process. Step one generates Treg cell progenitors (TregP) and is driven by strong TCR interactions with antigens presented in the thymus. Step two is initiated by activation of STAT5 via IL-2, or IL-15, leading to mature Treg cells capable of ...
    Fibroblast Gap-closure Assay-Microscopy-based in vitro Assay Measuring the Migration of Murine Fibroblasts
    Authors:  Agnieszka P. Looney and Mallar Bhattacharya, date: 08/20/2019, view: 350, Q&A: 0
    [Abstract] Pulmonary fibrosis is characterized by pathological scaring of the lung. Similar to other fibrotic diseases, scar formation is driven by excessive extracellular matrix deposition by activated, proliferative, and migratory fibroblasts.

    Currently, the two most widely used chemotaxis and cell migration assays are the scratch assay and the ...
    Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain
    Authors:  Hiroshi Yamaguchi and Luis de Lecea, date: 08/20/2019, view: 432, Q&A: 0
    [Abstract] Recently developed gene editing technologies based on engineered CRISPR/Cas9 systems enables researchers to disrupt genes in a cell type-specific manner in the adult mouse brain. Using these technologies, we recently showed that the dopamine beta-hydroxylase gene in Locus Coeruleus (LC) norepinephrine neurons plays a vital role in the maintenance ...
    A Novel Technique for Imaging and Analysis of Hair Cells in the Organ of Corti Using Modified Sca/eS and Machine Learning
    [Abstract] Here, we describe a sorbitol-based optical clearing method, called modified Sca/eS that can be used to image all hair cells (HCs) in the mouse cochlea. This modification of Sca/eS is defined by three steps: decalcification, de-lipidation, and refractive index matching, which can all be completed within 72 h. Furthermore, we established automated ...
    Three-dimensional Reconstruction and Quantification of Proteins and mRNAs at the Single-cell Level in Cultured Cells
    Authors:  Xueer Jiang, Ingrid Brust-Mascher and Li-En Jao, date: 08/20/2019, view: 512, Q&A: 0
    [Abstract] Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of ...

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