An automated screening platform for improving the responsiveness of genetically encoded Ca2+ biosensors in mammalian cells†
Abstract
Genetically-encoded, fluorescent protein (FP)-based biosensors are powerful tools for imaging dynamic cellular activities. Directed evolution is a highly effective method for developing enhanced versions of FP-based biosensors, but the screening process is laborious and time-consuming. Mammalian cell-based screening with electrical stimulation methods has been successful in accurately selecting variants of biosensors for imaging neuronal activities. We introduce an automated mammalian cell screening platform utilizing a fluorescence microscope and a liquid dispenser to enable the screening of biosensor responsiveness to chemical stimulation. We demonstrated the effectiveness of this platform in improving the response of a red fluorescent biosensor for Ca2+, K-GECO, for detection of histamine-induced changes in Ca2+ concentration. This method should be applicable to any FP-based biosensor that responds to pharmacological treatment or other exogenous chemical stimulation, simplifying efforts to develop biosensors tailored for specific applications in diverse biological contexts.