Calcium Flux Assays cAMP Assays

EMD Millipore′s Ready-to-Assay GPCR frozen cells are designed for simple, rapid calcium assays with no requirement for intensive cell culturing. EMD Millipore has optimized the freezing conditions to provide cells with high viability and functionality post-thaw. The user simply thaws the cells and resuspends them in media, dispenses cell suspension into assay plates and, following over night recovery, assays for calcium response.

EMD Millipore′s ChemiBrite cells co-express a GPCR along with a novel variant of clytin, a calcium-activated photoprotein, to enable sensitive luminescent detection of ligand-induced calcium flux. The ChemiBrite version of clytin contains a mutation that increases its affinity for calcium to a level that permits detection of cytosolic calcium in many cells with greater sensitivity than other mitochondrially expressed photoproteins. Luminescent calcium assays offer several advantages over fluorescent calcium assays including; lower substrate cost, increased sensitivity, and lack of interference from fluorescent compounds.

Follicular Stimulating Hormone (FSH) is a critical hormone in mammalian reproduction. In concert with Luteinizing Hormone (LH), this gonadotropin is secreted by the pituitary gland as a glycoprotein and affects all aspects of fertility and reproduction. In females, FSH drives ovarian development, maturation and cycle regulation. In males, FSH mediates spermatid maturation. FSH receptors couple to Gαs thus utilizing cAMP production to mediate cellular effects (Verrier et al). EMD Millipore′s cloned human FSH receptor-expressing ChemiBrite cells were made by stable transfection of HEK293 cells with ChemiBrite clytin, the FSH receptor (FSHR) and a promiscuous G protein to couple the receptor to the calcium signaling pathway, then cryopreserving the cells at an optimal time post-transfection. Upon thaw, recovery, and loading, the cells are ready for luminescent, fluorescent, and cAMP analysis of agonists, antagonists and modulators at the FSH receptor.

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