Applications Of The FLIPR Assay
The FLIPR assay can be used for several applications, including activation of ion channels, screening for ion channel agonists, validation of MrgD, and post-assay setup. This article will cover some of the common applications for this assay.
Activation Of Ion Channels
An activation of ion channels using a FLIPR assay allows researchers to screen a variety of compounds by measuring changes in the membrane potential. It is particularly useful for investigating the effect of agonists and antagonists on calcium and potassium channels. The test procedure uses a known agonist, Gq, in a concentration of 90 percent. The addition of the test compound reduces calcium mobilization by half.
Screening Ion Channel Agonists
The FLIPR(tm) assay is used to screen ion channel agonists, antagonists, and allosteric modulators. The assay detects calcium flux responses in the presence of agonists and antagonists. For agonists, test compounds are added to the assay chamber at 90% of their known concentration. Then, they are exposed to the known agonist.
Validation Of MrgD
This assay is based on the interaction of the extracellular half of the sixth transmembrane helix of MrgD with bulky hydrophobic residues of TM3 and TM6. These two residues interact with MrgD and form the active form. These structures provide a framework for designing MrgD drugs. They also serve as validation of a MrgD binding assay.
FLIPR assays measure intracellular calcium mobilization in cells. This method also measures the activation status of ion channels and GPCRs. It is applicable for cell lines, frozen cells, and suspension cells. FLIPR assays have a wide sensitivity range and large assay window. To perform a FLIPR assay, 100 cells are plated in each well of a 96-well plate.
In a recent study, the Flipr assay was successfully used to identify the potential of new clamping agents. This assay involves measuring the intracellular Ca2+ concentration with a fluorescent-based assay. This method has advantages over patch-clamp assays, which only measure intracellular Ca2+ concentration. Furthermore, the Flipr assay allows for direct comparison of the potency of compounds by comparing their peak amplitude and desensitization properties. In addition, it can validate subunit-specific compounds as well, a significant advantage over the conventional patch-clamp assay.