title: 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.
abstract: 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.
authors.affiliation: Screening Technologies and Pharmacology Department, Institut Pasteur Korea, Gyeonggi-do, South Korea.
authors.forename: Ok Ryul
authors.initials: OR
authors.lastname: Song
authors.forename: Tae-Hee
authors.initials: TH
authors.lastname: Kim
authors.forename: Xavier
authors.initials: X
authors.lastname: Perrodon
authors.forename: Changbok
authors.initials: C
authors.lastname: Lee
authors.forename: Hee Kyoung
authors.initials: HK
authors.lastname: Jeon
authors.forename: Zahir
authors.initials: Z
authors.lastname: Seghiri
authors.forename: Ho Jeong
authors.initials: HJ
authors.lastname: Kwon
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cechetto
authors.forename: Thierry
authors.initials: T
authors.lastname: Christophe
mesh_terms: 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
chemical_list: D001557:Benzenesulfonates; D002021:Buffers; D004396:Coloring Agents; D014867:Water; D049452:Green Fluorescent Proteins; C006796:brilliant blue; D004121:Dimethyl Sulfoxide