Mammalia

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    Protocols in Current Issue
    Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient
    [Abstract] The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or ...
    Characterization of Biological Motion Using Motion Sensing Superpixels
    [Abstract] Precise spatiotemporal regulation is the foundation for the healthy development and maintenance of living organisms. All cells must correctly execute their function in the right place at the right time. Cellular motion is thus an important dynamic readout of signaling in key disease-relevant molecular pathways. However despite the rapid ...
    A Cell Culture Model that Mimics Physiological Tissue Oxygenation Using Oxygen-permeable Membranes
    Authors:  Chloe-Anne Martinez, Peter A. Cistulli and Kristina M. Cook, date: 09/20/2019, view: 275, Q&A: 0
    [Abstract] Dissolved oxygen and its availability to cells in culture is an overlooked variable which can have significant consequences on experimental research outcomes, including reproducibility. Oxygen sensing pathways play key roles in cell growth and behavior and pericellular oxygen levels should be controlled when establishing in vitro models. ...
    In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay
    Authors:  G. Elif Karagöz, Jirka Peschek, Peter Walter and Diego Acosta-Alvear, date: 07/20/2019, view: 1103, Q&A: 0
    [Abstract] The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification ...
    Evaluation of Genotoxicity by Micronucleus Assay in vitro and by Allium cepa Test in vivo
    Authors:  Christina N. Banti and Sotiris K. Hadjikakou, date: 07/20/2019, view: 907, Q&A: 0
    [Abstract] The in vitro and in vivo genotoxicity of new metallodrugs either as Small Bioactive Molecules (SBAMs) or Conjugates of Metals with Drugs (CoMeDs) is evaluated by the micronucleus test and the Allium cepa assay, respectively. Fetal lung fibroblast cells (MRC-5), normal human corneal epithelial cells (HCEC) and ...
    Improved HTGTS for CRISPR/Cas9 Off-target Detection
    Authors:  Jianhang Yin, Mengzhu Liu, Yang Liu and Jiazhi Hu, date: 05/05/2019, view: 1072, Q&A: 0
    [Abstract] Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a “bait” DNA ...
    Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis
    Authors:  Koshi Imami and Tomoharu Yasuda, date: 04/20/2019, view: 1455, Q&A: 0
    [Abstract] Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and “clickable” amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling ...
    In vitro Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells
    [Abstract] The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating ...
    Quantitative Analysis of Cargo Density in Single-extracellular Vesicles by Imaging
    Authors:  Taketoshi Kajimoto and Shun-ichi Nakamura, date: 12/20/2018, view: 1522, Q&A: 0
    [Abstract] Function of extracellular vesicles such as exosomes and microvesicles is determined by their wide ranges of cargoes inside them. Even in the pure exosomes or microvesicles the cargo contents are very heterogeneous. To understand this heterogeneous nature of extracellular vesicles, we need information of the vesicles, which will give us some ...
    Electron Microscopic Detection of Proteins and Protein Complexes in Mammalian Cells Using APEX-tagged, Conditionally Stable Nanobodies
    Authors:  Thomas E. Hall, James Rae, Nicholas Ariotti and Robert G. Parton, date: 11/20/2018, view: 1557, Q&A: 0
    [Abstract] The recent development of 3D electron microscopic techniques for cells and tissues has necessitated the development of new methods for the detection of proteins and protein-complexes in situ. The development of new genetic tags, such as the ascorbate peroxidase, APEX, for electron microscopic detection of tagged proteins has expanded the ...



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