Mammalia

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    Protocols in Current Issue
    The RiboPuromycylation Method (RPM): an Immunofluorescence Technique to Map Translation Sites at the Sub-cellular Level
    Authors:  Amandine Bastide, Jonathan W. Yewdell and Alexandre David, date: 01/05/2018, view: 4775, Q&A: 0
    [Abstract] While isotopic labeling of amino acids remains the reference method in the field for quantifying translation rate, it does not provide any information on spatial localization of translation sites. The rationale behind developing the ribopuromycylation method (RPM) was primarily to map translation sites at the sub-cellular level while avoiding ...
    In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death
    Authors:  Yuting Ma and Heng Yang, date: 12/20/2016, view: 10579, Q&A: 1
    [Abstract] Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve ...
    Quantification of 2-Hydroxyglutarate Enantiomers by Liquid Chromatography-mass Spectrometry
    Authors:  William M. Oldham and Joseph Loscalzo, date: 08/20/2016, view: 6399, Q&A: 0
    [Abstract] Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al ...
    Wnt Reporter Activity Assay
    Author:  Chen Zhao, date: 07/20/2014, view: 22387, Q&A: 2
    [Abstract] This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt ...
    Hedgehog (Hh) Reporter Activity Assay
    Author:  Chen Zhao, date: 07/20/2014, view: 10353, Q&A: 0
    [Abstract] This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh ...
    In vitro Biomineralization Assay
    Authors:  Kyunghee Lee, Minsuk Kwon and Daewon Jeong, date: 02/05/2014, view: 12955, Q&A: 1
    [Abstract] Biomineralization in vertebrates has both physiological and pathological aspects. Physiological mineralization is essential for proper development and function of hard tissues, such as bone, teeth, and growth plate cartilage, but it does not occur in soft tissues. Pathological ectopic mineralization, in contrast, occurs in soft tissues, including ...
    ATP and Lactate Quantification
    Authors:  Isaline Rowe, Marco Chiaravalli and Alessandra Boletta, date: 12/05/2013, view: 11736, Q&A: 0
    [Abstract] Cells use glucose to generate energy by two different metabolic processes: lactic fermentation and aerobic respiration. In the first common series of reactions, glucose is converted into pyruvate. In anaerobic conditions, pyruvate is transformed into lactate, this process yields to 2 ATP molecules per glucose molecule. In the presence of oxygen, ...
    Determination of the Intracellular Calcium Concentration in Peritoneal Macrophages Using Microfluorimetry
    [Abstract] Calcium is one of the most important intracellular messengers in biological systems. Ca2+ microfluorimetry is a valuable tool to assess information about mechanisms involved in the regulation of intracellular Ca2+ levels in research on cells and in living tissues. In essence, the use of a dye that fluoresces in the presence ...
    Calcium Mobilisation Assay in Response to Chemokine Stimulation
    Author:  Darran G. Cronshaw, date: 05/05/2012, view: 11039, Q&A: 1
    [Abstract] This assay is used to measure calcium mobilization in lymphocytes (either in primary cells or cell lines) in response to chemokine stimulation using ratiometric analysis. It has also been used for measuring TCR mediated calcium flux. In addition, the same labeling procedure with the addition of brilliant black (a quenching agent) (Sigma-Aldrich, ...
    T Cell Calcium Mobilization Study (Flow Cytometry)
    Author:  Guo N. Huang, date: 05/05/2012, view: 11658, Q&A: 0
    [Abstract] Antigen recognition and activation of T cell receptor (TCR) triggers transient calcium release from intracellular compartments and subsequent sustained calcium influx through cell surface Icrac channels. Sustained elevation of the cytoplasmic calcium level activates many calcium-dependent enzymes and transcription factors, which are ...



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