Live Imaging Assay for Assessing the Roles of Ca<sup>2+</sup> and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Christina Tam; Andrew R. Flannery; Norma Andrews

doi:10.3791/50531

JoVE Journal
Biology

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JoVE Journal Biology

Live Imaging Assay for Assessing the Roles of Ca²⁺ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

实时成像检测方法的评估钙的作用 2 +和鞘磷脂酶在成孔毒素伤口的修复

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18:25 min

August 25, 2013

DOI: 10.3791/50531-v

Christina Tam¹, Andrew R. Flannery¹, Norma Andrews¹

¹Department of Cell Biology and Molecular Genetics,University of Maryland

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细胞暴露于所述亲脂性染料FM1-43的实时成像可以精确测定由该成孔毒素从质膜除去动力学。这是一个敏感的测定法可以用于评估要求的Ca ^{2 +，}鞘磷脂和其它因素对质膜修复。

该程序的总体目标是在活细胞成像中利用亲脂性染料 FM 1 43 来测量从质膜中去除性能不佳的毒素的精确动力学。这是通过首先在 4 摄氏度下预结合毒素来实现的。第二步是将培养皿转移到加热的显微镜载物台上，并找到一个可以观察细胞的焦平面。

接下来，添加适当的预热介质并立即开始成像。最后一步是分析和定量细胞内 FM 1 43 荧光。最终，这种灵敏的测定可用于评估钙、fingal、髓鞘和其他质膜修复因素的需求。

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