Intracellular characterization of the K+ indicator APG-1.

(A) Fluorescence image of primary astrocytes loaded using APG-1 AM. Scale bar 50 µm. (B) In situ excitation and emission spectra measured by fluorescence microscopy. Intracellular spectra were ∼10 nm red-shifted compared with measurement in cuvettes. (C) Representative experimental trace depicting the in situ calibration procedure. At the time indicated by the arrow, the cell membrane was permeabilized for K+ using valinomycin and nigericin while the Na+/K+ ATPase was inhibited by ouabain. Solutions of different [K+] were then sequentially applied until stable fluorescence plateaus were obtained. (D) Calibration curve obtained by plotting the fluorescence plateau values measured for each known [K+].