Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for identifying a rapid mixing method for high-content/throughput screening.
Rapid mixing in microplates is still an underappreciated challenge in screening assay development, particularly with the use of noncontact nanoliter liquid handlers. In high-content/throughput screening (HC/TS), fast and efficient mixing between compounds and cell culture medium is even more critical as biological kinetics dictates speed of mixing, usually within a few minutes. Moreover, mixing in HC/TS should be gentle enough to avoid any negative disruption in cell layer. Here the authors introduce a method to accurately quantify drop diffusion into a microplate well, independently of buffer, liquid handler, or dispensing protocol. This method was used to determine the effect of various mixing methods on the diffusion of a nanoliter drop of pure DMSO in aqueous buffer in 384-well plates. Rapid plate shaking and additional buffer addition were shown to be the most efficient and effective mixing methods for HC/TS. However, efficient mixing by plate shaking is limited by assay volume. Bulk addition shows fast and efficient mixing, without negative effects on cells. Moreover, this simple, fast, and inexpensive method can be easily adapted on any platform.
Song
Ok Ryul
OR
Screening Technologies and Pharmacology Department, Institut Pasteur Korea, Gyeonggi-do, South Korea.
Kim
Tae-Hee
TH
Perrodon
Xavier
X
Lee
Changbok
C
Jeon
Hee Kyoung
HK
Seghiri
Zahir
Z
Kwon
Ho Jeong
HJ
Cechetto
Jonathan
J
Christophe
Thierry
T
D001557:Benzenesulfonates / Q000378:metabolism; D002021:Buffers; D002460:Cell Line; D002498:Centrifugation; D004396:Coloring Agents / Q000378:metabolism; D004058:Diffusion; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods*; D018613:Microscopy, Confocal; D009038:Motion; D015203:Reproducibility of Results; D013997:Time Factors; D014867:Water / Q000737:chemistry
D001557:Benzenesulfonates; D002021:Buffers; D004396:Coloring Agents; D014867:Water; D049452:Green Fluorescent Proteins; C006796:brilliant blue; D004121:Dimethyl Sulfoxide