The FLIPR® System

Membrane Potential Assays

 
The FLIPR® system was originally developed for Dr. Vince Groppi of Pharmacia-Upjohn. Dr. Groppi had developed an assay using the voltage sensitive dye DiBac and a confocal imaging system, however, his system had limited throughput. The FLIPR system was designed to perform the assay in a 96-well format to allow for high-throughput screening. The difficult aspect of this particular assay is the strong background component to the fluorescence data, making it necessary to be able to discriminate between the fluorescence signal from a cell monolayer and the background fluorescence from the extracellular medium. The FLIPR system's patented optical design was developed to do exactly that.

		Figure 1. The effect of various compounds on A10 cells’ membrane potentials.

Figure 1 represents the results of a typical membrane potential assay run in the FLIPR system. In this example, Al0 smooth muscle cells were grown to near confluence in a microplate, stained with DiBAC, then tested in the FLIPR system. Due to the presence of endogenous potassium channels in the cells, adding 30 mM extracellular potassium causes a membrane depolarization, and thus an increase in fluorescence. At the same time, other wells in the plate were stimulated with a well-known potassium channel opener (1 mM Pl075). This effectively hyperpolarizes the membrane and causes a reduction in fluorescence. Some of the cells were also pre-incubated with various concentrations of glyburide, a known potassium channel blocker. As shown, partial and complete blocking of the hyperpolarization resulted from exposure to 1 µM and 10 µM concentrations of glyburide, respectively. The dye has been proven to be linear over physiological ranges from -100 to 100 mV, and the typical detection limit in the FLIPR system is approximately 5 mV. The limit will vary slightly according to the cell type used.

membrane potential on the FLIPR system

The data in Figure 2 were generated using THP-1 cells loaded with 5 µM DiBac and dispensed into a 384-well microplate. These plates were run on the FLIPR³⁸⁴ system using the heated stage. The cells were exposed to either a depolarizing concentration of KCl (30 mM) or to a hyperpolarizing concentration of the purinergic receptor agonist UTP (10 µM). KCl-mediated depolarization resulted in increased fluorescence, while the UTP-mediated hyperpolarization resulted in decreased fluorescence. These data are an overlay of representative wells for each condition.

		Figure 2. The effect of KCl and UTP on THP-1 cells.