Quantitative Cell-Based Reporter Gene Assays Using Droplet-Based Microfluidics

被引：77
作者：
Baret, Jean-Christophe [1 ]
Beck, Yannick [1 ,2 ]
Billas-Massobrio, Isabelle [2 ]
Moras, Dino [2 ]
Griffiths, Andrew D. [1 ]
机构：
[1] Univ Strasbourg, CNRS, UMR 7006, ISIS, F-67083 Strasbourg, France
[2] IGBMC, Dept Biol & Genom Struct, F-67400 Illkirch Graffenstaden, France
来源：
CHEMISTRY & BIOLOGY | 2010年 / 17卷 / 05期
关键词：
ECDYSONE RECEPTOR; SINGLE CELLS; ENCAPSULATION; LIBRARIES; SYSTEM; MICROCHANNELS; MICRODROPLETS; AMPLIFICATION; ULTRASPIRACLE; EVOLUTION;
D O I：
10.1016/j.chembiol.2010.04.010
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
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.
引用
收藏
页码：528 / 536
页数：9
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