Measuring Ca²⁺ changes in multiwell format using the Fluorometric Imaging Plate Reader (FLIPR(®))

The Fluorometric Imaging Plate Reader (FLIPR) has made a significant contribution to drug discovery programs. The key advantage of FLIPR over conventional plate readers is the ability to measure fluorescence emission from multiple wells (96 wells or 384 wells) simultaneously and with high temporal resolution. Consequently, FLIPR has been used extensively to record dynamic intracellular processes such as changes in intracellular Ca(2+) ion concentration, membrane potential, and pH. Since FLIPR is used to measure a functional response in cells, it is rapidly able to distinguish full agonists, partial agonists, and antagonists at a target of interest, making the system a valuable screening tool for interrogation of compound libraries. Automated FLIPR systems for ultra high throughput have also become available that employ integrated plate stackers, washers and specialized stages to allow users to shuttle cell and compound plates from incubators or storage magazines onto the FLIPR system itself. In this chapter generic methods for assessing intracellular Ca(2+) on the FLIPR are described.