Abstract: AimTo discover new regulators of potassium channel, an in vitro assay based on DiBAC₄(3) to determine the fluorescence was established for high throughput screening. MethodsA cell-based 96-well format fluorescence assay using DiBAC₄(3) in cultured PC12 cells was described. Cells were loaded with 5 μmol·L^-1 DiBAC₄(3) and incubated at 37 ℃ for 30 min before adding KCl or several known potassium channel regulators. The cellular DiBAC₄(3) fluorescence responce was then detected. The fluorescence changes can be used to evaluate membrane potential changes, which are determined mainly by potassium channels. Results Extracellular high K⁺-induced depolarization and several potassium channel blockers including 4-AP, TEA, E-4031, glibenclamide, quinidine and nifedipine all evoked increases in DiBAC₄(3) fluorescence responce. The potassium channel opener, cromakalim, evoked decrease in DiBAC₄(3) fluorescence responce. The fluorescence changes of 4-AP, TEA, glibenclamide, nifedipine and cromakalim were in a concentration-dependent manner. In 76 compounds screened by using the established DiBAC₄(3)-based assay, 9 compounds were found to change the fluorescence dose-dependently. Patch clamp technique is needed to further testify and screen their actions on potassium currents. ConclusionThe DiBAC₄(3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulators.