Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement
Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement
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DOI：
10.2144/00291st02
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发表时间：
2000-07-01
期刊：
影响因子：
2.7
通讯作者：
Theodorescu, D
中科院分区：
文献类型：
--
作者：
Gildea, JJ;Harding, MA;Theodorescu, D
目前用于评估肿瘤运动性的体外试验可以通过开发一种简单的技术来改进，该技术将有助于研究特定基因对肿瘤改变的趋化性的影响。我们开发了一种技术，通过使用荧光和发光来评估趋化性，改进了经典的transwell测定法。在该瞬时转染系统中，将报告构建体和对运动性具有未知影响的基因的共转染与生化测定偶联，以定量已接受转移基因的细胞的数量，所述转移基因随后穿过膜。发现该测定比常规transwell室的可变性更小，并且容易适用于细胞运动或细胞侵袭的研究。我们还证明，该试验可以检测单独和组合的遗传和药理学抑制运动的效果。因此，它有可能显示出累加或协同效应。
Current in vitro assays used in assessing tumor motility could be improved by the development of a simple technique that would facilitate studies of the impact of specific genes on pharmacologically altered chemotaxis. We developed a technique that improves on the classic transwell assay by using fluorescence and luminescence to assess chemotaxis. In this transient transfection system, co-transfection of a reporter construct and a gene with an unknown impact on motility are coupled with biochemical assays to quantitate the number of cells that have received a transferred gene, which subsequently, crosses the membrane. This assay was found to be less variable than the conventional transwell chamber and is easily adaptable to studies of cell motility or cell invasion. We also demonstrate that this assay can detect the effect of both genetic and pharmacological inhibition of motility alone and in combination. It therefore has the potential to reveal additive or synergistic effects.