Both Gq-coupled and non-Gq coupled GPCRs are tested for their functional expression using the FLIPR Calcium 4 Assay Kit. Non Gq-coupled GPCRs are co-expressed with promiscuous Gα16 or chimeras Gqi5. Below is a detailed protocol describing calcium flux measurements using Flexstation (Molecular Devices).

A. Preparation of Cells

1. Culture adherent cells in 96- or 384-well microplates (black with clear bottom) overnight to near confluence.

B. Preparation of Reagents

1. Make a 250 mM (100X) stock solution of probenecid acid (Invitrogen P36400).
2. Prepare the dye loading solution: add 10 ml of Assay Buffer (see Notes below) and 100 µl of probenecid acid stock solution to a vial of dye loading mix (provided in the kit). Vortex to completely dissolve the solid.

C. Assay

1. Add equal volume of the dye loading solution to each well containing cells in medium (or Assay Buffer). Incubate the cells at 37 °C, 5% CO₂ for 1 hour.
2. During the incubation, prepare a solution of receptor agonist (5X) in Assay Buffer. Make serial dilution in a compound plate (Nunc 442587).
3. Measure fluorescence using Flexstation 1 hour after the addition of dye mixture to the cells. Instrument settings: excitation at 485 nm, emission at 525 nm, cut-off at 515 nm, assay duration 90 seconds (or longer depending on receptor). Adjust injection speed, volume and pipette height based on plate format and cell status.

Notes:

1. Assay Buffer: routinely we use Hanks’ Balanced Salt Solution with 20 mM HEPES, pH 7.4. Some receptors/ligands require PBS or 0.9% NaCl.
2. Some receptors need starvation prior to assay.
3. Cells that attach to the plates loosely (such as HEK293) require poly-D-lysine or collagen coated microplates.
4. Adjust volumes if using antagonists or modulators.
5. For further details, please see FLIPR Calcium 4 Assay Kit Downloadable Literature.