GPCR CALCIUM PRODUCT SOLUTIONS

Calcium No Wash^PLUS Detection Kit

• Homogeneous single step, no wash HTS friendly assay protocol
• Detect calcium mobilization in adherent or suspension cells for either GPCRs or calcium ion channels
• Large assay windows, wide dynamic range, and improved signal and performance over FLUO-3
• Multiplex with β-arrestin recruitment assays using the same cell line and the same well for multiple readouts

Calcium Gq-coupled Cell Lines

• Native cell lines overexpressing naturally Gq-coupled, wild-type GPCRs
• Optimized for use with Calcium No Wash^PLUS assay for detection of calcium mobilization in response to an agonist stimulation

ChemiSCREEN™ Cell Lines

• Cell lines containing a promiscuous G protein, Gα15, to help funnel signaling to a common calcium readout, regardless of G protein coupling status
• High functional expression of receptors with the ability to assay both agonist and antagonist in a single well
• Stable cell lines for continuous culture with increased reliability and reproducibility assay results
• Perform orthogonal assay confirmation such as second messenger signaling, e.g. cAMP, and ERK phosphorylation, for most targets

ChemiBRITE™ Cell Lines

• Cell lines modified to flash with added brightness upon GPCR activation
• Exceptional high signal-to-background ratio with no assay interference with auto-fluorescent compounds
• Tool box capabilities allows generation of your own stable cell lines

ChemiSCREEN™ and ChemiBRITE™ Frozen Cells

• Ready-to-assay GPCR frozen cells come with plating media and enough cells to run either 96- or 384-well plate assays in full or half plate formats
• Qualified for cAMP and calcium second messenger assays with results within 24 hours to accelerate your data generation

Calcium No Wash^PLUS Assay Principle

The calcium G protein-dependent pathway involves a heterotrimeric (α/β/γ) G-protein containing a GDP molecule (not shown here) bound to the Gα subunit, which holds the trimer together. Upon activation, GDP is exchanged for GTP, leading to the dissociation of the Gβ/Gγ dimer from Gα. Both parts remain anchored to the membrane and become free to act upon their downstream effectors and initiate unique intracellular signaling responses. The activated Gα subunit interacts with and regulates many effector molecules such phospholipase C (PLC) that can ultimately lead to the release of calcium (a second messenger) from the ER.

The Calcium No Wash^PLUS Detection Kit is calcium mobilization, cell-based assay which uses an esterified (inactive) calcium dye (probe) that penetrates the cell membrane and becomes active once inside the cell. The active form of the dye becomes fluorescent after binding to intracellular calcium. Additive A is added to prevent the dye from being released out from the cell. Ligand binding stimulates GPCR activation, resulting in the release of intracellular calcium stores from the ER, which leads to an increase in fluorescence in the presence of the activated calcium dye.

Perform Rank Order Analysis of Potential Leads

Dose response and rank order potency for calcium mobilization and β-arrestin recruitment assays using the same cell line. The PathHunter CHO-K1 MRGPRX2 β-Arrestin Cell Line (Cat. No. 93-0309C2) was used to analyze both calcium mobilization with the Calcium No Wash^PLUS detection kit and β-arrestin recruitment.

– Readouts showing select agonists effect on (A.) calcium mobilization of intracellular calcium and on (B.) β-arrestin recruitment upon binding to MrgprX2 receptors. Both dose response curves are the mean of two replicates and show normalized data calculated from the maximum effect obtained with cortistatin-14. 

– C. Table showing potency and efficacy of agonist relative to cortistatin-14. Results show good agreement with those previously published with respect to rank orders of potency agonists (Grimes et al. 2019) maintained relative to cortistatin-14 used as reference agonist for both assays.