Quantitative Cell-Based Reporter Gene Assays Using Droplet-Based Microfluidics
Quantitative Cell-Based Reporter Gene Assays Using Droplet-Based Microfluidics
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DOI：
10.1016/j.chembiol.2010.04.010
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发表时间：
2010-05-28
影响因子：
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通讯作者：
Griffiths, Andrew D.
中科院分区：
文献类型：
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作者：
Baret, Jean-Christophe;Beck, Yannick;Griffiths, Andrew D.
我们使用基于液滴的微流体系统对核受体配体进行基于定量的细胞报告基因测定。单个BOMBYX MORI细胞在纳米液滴中被划分为小液滴，该液滴的功能比微量磁板较小> 1000倍的微型反应器，以及八个或十个离散浓度的20-羟基丁香酮，由30年的芯片稀释产生，并在3年中产生，并且由荧光标签编码。记者基因和荧光标签的绿色荧光蛋白表达的同时测量允许在单细胞水平上构建激素的剂量反应谱。与每个浓度相似的筛查类似于7500个单元，提供了统计相关的数据，可精确测量EC50（70 nm +/- 12％，alpha = 0.05），与标准方法以及文献数据一致。
We used a droplet-based microfluidic system to perform a quantitative cell-based reporter gene assay for a nuclear receptor ligand. Single Bombyx mori cells are compartmentalized in nanoliter droplets which function as microreactors with a >1000-fold smaller volume than a microtiter-plate well, together with eight or ten discrete concentrations of 20-hydroxyecdysone, generated by on-chip dilution over 3 decades and encoded by a fluorescent label. The simultaneous measurement of the expression of green fluorescent protein by the reporter gene and of the fluorescent label allows construction of the dose-response profile of the hormone at the single-cell level. Screening similar to 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC50 (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data.