• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Measurement of Ca2+ Fluxes in Permeabilized Cells Using 45 Ca2+ and Fluo-3

        互联网

        1040
        Many cell-surface receptors via G-proteins activate phosphoinositidase C, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce the second messengers, myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 or IP3 ] and diacylglycerol (1 ). Ins(1,4,5)P3 interacts with a specific receptor populations of ligand-gated channels to mobilize nonmitochondrial intracellular Ca2+ stores (1 ). Because Ins(1,4,5)P3 is plasma-membrane-impermeant, this phenomenon was first demonstrated in permeabilized pancreatic acinar cells (2 ), and all subsequent studies in cells have involved introduction of Ins( 1,4,5)P3 by rendering a cell population permeable (3 ), using microinjection techniques (4 ), or by the presentation of chemically modified membrane-permeable Ins(l,4,5)P3 analogs, such as photolabile “caged-IP3 ” (5 ). An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca2+ stores (6 , 7 ). However, these preparations exhibit a loss of Ins(1,4,5)P3 -responsiveness compared to cells. Here we describe a 45 Ca45 -release assay and a fluo-3 assay, two methods we use to monitor Ins(l,4,5)P3 -induced Ca2+ mobilization from nonmitochondrial intracellular Ca2+ stores using cytosol-like buffer (CLB) and permeabilized SH-SY5Y neuroblastoma cell populations. The 45 Ca2+ -release assay was similar to those previously described (3 , 8 ), and involves preloading a “spike” of 45 Ca2+ into the intracellular Ca2+ stores of permeabilized cells and then monitoring the resultant release of 45 Ca2+ induced by concentrations of Ins(1,4,5)P3 and other agonists. We have utilized this assay to assess the intrinsic activity of Ins(1,4,5)P3 a wide range of Ins(l,4,5)P3 analogs, and other Ca2+ -mobilizing agents. The assay can be undertaken at a low cell density to minimize cellular metabolism of the inositol polyphosphates, or at high cell density to allow metabolic time-courses to be associated with Ca2+ mobilization.
        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序