Section10:Types of Flipr™ formats

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GPCR Targets Coupled to Ca+2 Mobilization

GPCR targets that naturally couple via Gq produce a ligand-dependent increase in intracellular Ca+2 that can be measured using a calcium-sensitive dye. GI/o-coupled GPCR receptor activation can be “switched” to induce an increase in intracellular calcium in two ways: by the use of chimeric G-proteins (Gαqi5 or Gαqo5), or by engineering the cells to over-express a promiscuous G-protein (G α16 or Gα15).

The integrated pipettor capabilities of the FLIPRTM, as well as internal software modifications, provide an opportunity to detect agonists, antagonists, and allosteric modulators all in one assay. One-, two-, or three-addition assays may be performed depending on the desired assay format. A one-addition assay can be performed to detect agonists, where the compound of interest is added to look for a response. This mode could also be used to look for allosteric modulators or antagonists if the test compounds are added “off-line”, although this is not the preferred method of operation. Until 2006, the two-addition assay was the standard assay format. In this method, the test compounds are added in the presence of an EC10 dose of the agonist in the first addition to detect agonists or allosteric modulators. The second addition is an EC90 dose of the max control to identify antagonists. While this scheme works, it requires a secondary assay to distinguish the agonists from the allosteric modulators; this need was abolished by the advent of a three-addition assay. In the three-addition mode, you can detect all three modes of activity in a single assay, saving considerable time and reagents. Another advantage found during testing of the three-addition assay was better mixing and a pre-incubation of the cells with compound resulting in better identification of potentiators. Typical assay formats and the resulting curves are summarized below (Table 1).

Ion Channels with Significant Ca2+ Permeability

Ion channel targets with significant Ca+2 permeability, such as the iGluRs, produce an increase in intracellular calcium that can be measured using calcium-sensitive dyes and the FLIPR™ instrument. The methodology used is analogous to that for the GPCRs.

Ion Channels which Produce Significant Changes in Membrane Potential

Ion channel targets such as the iGluRs with ion permeability that significantly affects the membrane potential can be measured using a membrane potential dye and the FLIPR™ (see Baxter, et al).