Quantitative cell-based reporter gene assays using droplet-based microfluidics.
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 approximately 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC(50) (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data.
Institut de Science et d'Ingénierie Supramoléculaires, Université de Strasbourg, CNRS UMR 7006, 8 Allée Gaspard Monge, BP 70028, F-67083 Strasbourg Cedex, France. jc.baret@unistra.fr
Jean-Christophe
JC
Baret
Yannick
Y
Beck
Isabelle
I
Billas-Massobrio
Dino
D
Moras
Andrew D
AD
Griffiths
D000818:Animals; D012831:Bombyx / Q000378:metabolism; D004441:Ecdysterone / Q000378:metabolism; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000379:methods*
D049452:Green Fluorescent Proteins; D004441:Ecdysterone