Mammalia

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    Protocols in Current Issue
    Mitochondrial Respiratory Measurements in Patient-derived Fibroblasts
    Authors:  Prashant Mishra, Ting Zhang, Ming Guo and David Chan, date: 12/05/2019, view: 67, Q&A: 0
    [Abstract] Mitochondrial dysfunction is associated with a number of human diseases. As an example, we recently established in vivo Drosophila models of IBMPFD (Inclusion body myopathy, Paget disease, and frontotemporal dementia), and uncovered that human disease mutations of the p97/VCP (Valosin Containing Protein) gene behave as ...
    Enhanced-ice-COLD-PCR for the Sensitive Detection of Rare DNA Methylation Patterns in Liquid Biopsies
    Authors:  Florence Mauger and Jörg Tost, date: 12/05/2019, view: 66, Q&A: 0
    [Abstract] In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, ...
    An Optimized Method to Isolate Human Fibroblasts from Tissue for Ex Vivo Analysis
    [Abstract] Despite their involvement in many physiological and pathological processes, fibroblasts remain a poorly-characterized cell type. Analysis of primary fibroblasts while maintaining their in vivo phenotype is challenging: standard methods for fibroblast isolation require cell culture in vitro, which is known to alter phenotypes. ...
    Proximity Ligation Assay for the Investigation of the Intramolecular Interaction of ELMO1
    Authors:  Wai Wa Ray Chan, Dik Long Dennis Chau, Wen Li and Kwok-Fai Lau, date: 12/05/2019, view: 93, Q&A: 0
    [Abstract] Intramolecular interaction is a common mechanism that regulates protein activities. Conventionally, such interactions are investigated by classical in vitro biochemical assays. Here, we describe a protocol for studying the intramolecular interaction of cell motility and engulfment 1 (ELMO1) in mammalian cells by using proximity ligation ...
    DNase I Chromatin Accessibility Analysis
    Authors:  Brook S. Nepon-Sixt and Mark G. Alexandrow, date: 12/05/2019, view: 78, Q&A: 0
    [Abstract] Chromatin consists of compacted DNA in complex with proteins and contributes to the organization of DNA and its stability. Furthermore, chromatin plays key roles in regulating cellular processes such as DNA replication, transcription, DNA repair, and mitosis. Chromatin assumes more compact (inaccessible) or decondensed (accessible) conformations ...
    Mapping the Mechanome–A Protocol for Simultaneous Live Imaging and Quantitative Analysis of Cell Mechanoadaptation and Ingression
    [Abstract] Mechanomics, the mechanics equivalent of genomics, is a burgeoning field studying mechanical modulation of stem cell behavior and lineage commitment. Analogous to mechanical testing of a living material as it adapts and evolves, mapping of the mechanome necessitates the development of new protocols to assess changes in structure and function in ...
    SarkoSpin: A Technique for Biochemical Isolation and Characterization of Pathological TDP-43 Aggregates
    Authors:  Manuela Pérez-Berlanga, Florent Laferrière and Magdalini Polymenidou, date: 11/20/2019, view: 535, Q&A: 0
    [Abstract] TDP-43 is the main aggregating protein in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Aggregated TDP-43 is resistant to diverse detergent solubilization, yet physiological TDP-43 and other abundant proteins commonly co-purify with pathological TDP-43. This mixed isolation has ...
    Opto-magnetic Selection and Isolation of Single Cells
    Authors:  Loïc Binan, Joannie Roy and Santiago Costantino, date: 11/20/2019, view: 337, Q&A: 0
    [Abstract] Capturing single cells from large heterogenous populations based solely on observable traits is necessary for many cell biology applications and remains a major technical challenge. The protocol we present allows the isolation of viable and metabolically active cells selected for their shape, migration speed, contact to other cells, or ...
    Imaging VIPER-labeled Cellular Proteins by Correlative Light and Electron Microscopy
    [Abstract] Advances in fluorescence microscopy (FM), electron microscopy (EM), and correlative light and EM (CLEM) offer unprecedented opportunities for studying diverse proteins and nanostructures involved in fundamental cell biology. It is now possible to visualize and quantify the spatial organization of cellular proteins and other macromolecules by FM, ...
    Implementing VIPER for Imaging Cellular Proteins by Fluorescence Microscopy
    Authors:  Julia K. Doh, Caroline A. Enns and Kimberly E. Beatty, date: 11/05/2019, view: 260, Q&A: 0
    [Abstract] Genetically-encoded tags are useful tools for multicolor and multi-scale cellular imaging. Versatile Interacting Peptide (VIP) tags, such as VIPER, are new genetically-encoded tags that can be used in various imaging applications. VIP tags consist of a coiled-coil heterodimer, with one peptide serving as the genetic tag and the other (“probe ...



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