Section10:Introduction

From Assay Guidance Wiki

The introduction of FLIPR™ (Fluorescence Imaging Plate Reader) in the 1990's provided biologists with a fast and easy method of detecting GPCR activation through changes in intracellular calcium concentration. By coupling receptors to Gq proteins which stimulate intracellular calcium flux upon binding, a functional response can be measured using calcium-sensitive dyes and a fluorescence plate reader. The FLIPR™ instrument has a cooled CCD camera imaging system which collects the signal from each well of a microplate simultaneously. The FLIPR™ can read at sub-second intervals, which enables the kinetics of the response to be captured, and has an integrated pipettor that may be programmed for successive liquid additions.

The integrated pipettor capabilities of the FLIPR™ provide an opportunity to detect agonists, antagonists, and allosteric modulators of GPCRs all in one assay. In the first addition, compounds of screening interest are added. The timing can be adjusted to allow for a pre-incubation period with the compounds, and agonist activity is detected by monitoring the calcium flux response in this step. In the second addition, a small amount of a known agonist that results in ~10% of maximal response is added to detect potentiator activity. The third addition consists of a maximal concentration of known agonist (~90% of the maximal response) to test for antagonism. This experimental design can encompass either two or three additions depending on the specific responses to be detected.

The FLIPR™ has also been utilized to screen ion channel targets using membrane permeable fluorescent dyes, such as the bis-oxanol dye DiBAC4(3), to measure changes in membrane potential. Compared to the rapid sub-second kinetics of channel opening observed by electrophysiology approaches, redistribution of the dye often takes minutes to produce a measurable response, and has prompted the development of more rapid dyes compatible with the FLIPR™.