( medium with 10% fetal bovine serum 1%...)Ca++-Induced G Protein Coupled Receptor (GPCR) Activation Monitored via Aequorin Luminescence in the LMax Microplate Luminometer (MaxLine Application Note #42)

2. The day before the experiment, cells in growth medium as above were seeded in 96-well microplates at a density of 60,000 cells/well and incubated overnight in a 37C, 5% CO₂ incubator.

3. Cells were loaded with coelenterazine by aspirating the growth medium from the wells and replacing it with 100 L coelenterazine loading solution/well. They were then incubated at 37C with 5% CO₂ for an additional 2 hours.

ASSAY PROCEDURE
1. A dilution series of UTP was prepared in H&H with 1% BSA concentrations ranging from 0.2 M to 200 M, with the diluent (0 M UTP) as the blank. All were placed in 15-mL conical tubes.

2. The plate containing the coelenterazine-loaded cells (100 uL/well) was placed into the drawer of the Lmax.

3. The SOFTmax PRO for Lmax was set up to inject 100 L of UTP reagent through the M injector and to begin reading immediately and take 20 consecutive 1-second reads on selected replicate wells.

4. Starting with the blank (0 M UTP in diluent) the M injection system was sequentially filled with increasing concentrations of UTP solutions by pumping 1.5 mL through it and then the selected replicate target wells for each concentration were read. The UTP-containing tubes were placed in a small rack on top of the Lmax so that the injector inlet tubing could easily be moved from one tube to the next higher concentration after each set of measurements was complete.

REACTION PROFILES AND DOSE/RESPONSE RESULTS
The reaction profiles of coelenterazine(h)-loaded cells to 0.1 - 3 uM UTP (final concentrations) are shown in Figure 2. When CHO cells expressing apo-aequorin protein (CHOMQ6 cells) were incubated with 2.5 uM coelenterazine(h), then exposed to UTP (a potent purinergic agonist), they responded
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