Section10:Potential Artifacts

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Although the FLIPR™ has facilitated advances in cellular calcium mobilization screens, these assays remain difficult to configure, relatively slow, and fraught with potential artifacts. Blocked FLIPR™ tips will lead to false positives in an inhibitor screen, or false negatives in an agonist screen. Fluorescent compounds, Ca+2 ionophores, and compounds that permeabilize the cell membrane can all contribute to false positives in the agonist read (Figure 11). These types of nuisance or interference compounds can often be identified from the kinetic traces of the response, but this kind of in depth data review is time consuming and requires experience to correctly recognize strange response profiles. In addition, compounds with agonist activity may interfere with antagonist reads due to desensitization or internalization of the receptor, resulting in false positives.

The utility of the FLIPR™ and calcium dye approach for screening GPCR targets has been greatly enabled by the use of over-expression of promiscuous and chimeric G proteins that provide a method to “switch” GI/o-coupled receptor activation to an increase in intracellular calcium. However, screens designed to detect receptor activity against a backdrop of stable, high-level promiscuous G protein expression are also susceptible to artifacts - - false positives derived presumably from other cell surface receptors hi-jacking the promiscuous G proteins. Indeed, even in the absence of a promiscuous G-protein, any endogenous GPCR that couples through Gq and induces a Ca+2 response may show up as an agonist or interfere with antagonist reads. It is well documented that GPCRs, particularly those in heterologous expression systems, can activate multiple signal transduction pathways, and indeed there is also evidence for cross-talk between recombinant and native receptors that may also complicate the responses to compounds. Thus, we recommend routinely performing a secondary screen against the parent cell line that lacks the receptor of interest in order to definitively identify false positives.