Fluorescence-Based High-Throughput Functional Profiling of Ligand-Gated Ion Channels at the Level of Single Cells
HEK293 cells were transiently co-transfected with YFPI152L and GlyR cDNA (). Approximately 48 h later, cells were seeded into the wells of 384-well plates at defined density and are cultured for another 24 h (). Functional analysis of GlyRs is carried out by progressive receptor activation and iterative fluorescence imaging using an in house-built automated screening device with integrated liquid-handling robotics (). Recorded images are segmented and fluorescence dose-responses calculated () are fitted (). Finally, functional parameters measured in single cells, such as R2, ΔF, slope and EC50 are filtered to discriminate functional from non-functional data ().