cAMP Assay Protocol

Following ligand binding, GPCR activation sets in motion a series of second messenger cascades that result in a physiological response. Adenylate cyclase, a membrane-bound enzyme, is involved in the signaling process. Ga_s- and Ga_i-coupled receptors block or stimulate adenylate cyclase, respectively, to regulate cAMP. The cAMP Assay allows cells that overexpress GPCRs to monitor the activation of Ga_s- and Ga_i-coupled receptors by using the GPCR's inherent coupling status.

Fig.1. Schematic diagram showing G protein-coupling mechanisms. (Sharan & Catherine, 2017)

Cell Thawing

1. Take the vial out of the liquid nitrogen tank and immediately defrost the cells in a 37°C water bath.

2. Decontaminate the outside of the vial with 70% ethanol just before the cells are totally frozen, then transfer the cells to a 15 mL centrifuge tube containing 9 mL complete growth media.

3. Remove the medium and pellet the cells by centrifugation at 200g force for 5 minutes.

4. Rehydrate the cells by resuspending them in full growth medium.

5. Pour 10 mL of complete growth media into a 10 cm dish with the cell suspension.

6. Incubate the cells at 37°C with 5% CO₂ in an incubator.

7. The next day, add the antibiotic.

Cell Subculturing

1. Take the culture medium out of the cells.

2. Remove all remnants of serum containing trypsin inhibitor by washing cells in PBS (pH 7.4).

3. In a 10 cm plate, pour 2.0 mL of 0.05% (w/v) Trypsin-EDTA solution and examine the cells under an inverted microscope until the cell layer has dispersed (usually within 3 to 5 minutes).

Note: Do not disturb the cells by hitting or shaking the dish while waiting for them to detach to avoid clumping. If the cells are hard to separate, place the dish in a 37°C incubator for 2 minutes.

4. Gently pipette 6.0 to 8.0 mL of full growth media into the plate to aspirate the cells.

5. Remove the medium and centrifuge the cells for 5 minutes at 200g force.

6. Re-suspend the cells in culture medium and transfer to a fresh culture dish.

7. Incubate the cells at 37°C with 5% CO₂ in an incubator.

cAMP Assay

When a ligand binds to receptors, G-protein signaling pathway is activated, leading to a cellular response mediated by cAMP. The cAMP signal can be monitored by measuring cAMP concentrations using cAMP detection kit. Please refer to the manufacturer's detection product user manual for protocols for cAMP measurement.

Reference

1. Sharan, S., Catherine, H. Potential of GPCR-Targeting Insecticides for Control of Arthropod Vectors: Volume 2: GPCRs and Ion Channels. 2017.