Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Sylvia T. Cheung; Soroush Shakibakho; Eva Y. So; Alice L-F Mui

doi:10.3791/52807

JoVE Journal
Biology

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JoVE Journal Biology

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

转染RAW264.7细胞用荧光素酶报告基因

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12:58 min

June 18, 2015

DOI: 10.3791/52807-v

Sylvia T. Cheung^1,2,3, Soroush Shakibakho^1,2, Eva Y. So^1,2, Alice L-F Mui^1,2,3

¹Immunity and Infection Research Centre,Vancouver Costal Health Research Institute, ²Department of Surgery,University of British Columbia, ³Department of Biochemistry and Molecular Biology,University of British Columbia

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由于细胞对外来物质的自然反应，转染到巨噬细胞系 RAW264.7 中是困难的。我们在这里描述了一种将荧光素酶报告基因转染到 RAW264.7 细胞中的温和而强大的程序。

该程序的总体目标是在不激活细胞的情况下将荧光素酶报告构建体转染到原始 2 6 4 0.7 细胞中。这是通过首先通过轻轻移液从储备板中收获原始 2 6 4 0.7 细胞并将细胞接种到 24 孔组织培养处理板中来实现的。第二步是 cot 转染质粒的 Firefly 荧光素酶报告，对照 RAN 荧光素酶质粒进入细胞。

这些质粒需要不含内毒素。接下来，用所需的处理刺激细胞，然后进行细胞裂解并转移到底部透明的白色 96 孔板中。最后一步是测定萤火虫荧光素酶活性，然后运行荧光素酶活性以计算萤火虫与比率。

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