Fig. 1

Experimental design for assessing the chloride extrusion activity of the K–Cl cotransporter KCC2 in neuronal cells. a Schematic depiction of the Cl-sensor. Cl-sensor consists of a triple YFP mutant fused via a short 20 amino acid linker with the CFP protein. b Schematic picture of the inverted fluorescence imaging setup used in the study. Grown on a coverslip, Neuro-2a cells were perfused with extracellular solution (ECS) in the absence and then in the presence of furosemide. Combination of high [K⁺] and glycine (with and without furosemide) was applied via the local perfusion system during the chloride-loading step. Details of the excitation and emission filters are described in the “Methods” section. c Representative optic view of Neuro-2a cells consequently excited with the 430 and 500 nm light. Regions of interest (ROIs) used for the subsequent analysis of the emitted fluorescence intensities for this optic view are indicated by yellow circles