Table 1.
cAMP Nucleotide-Gated Channel cAMP Biosensor Assay
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Plate cells | 20 μL | 10,000 cells per well |
| 2 | Incubation | 18 h | 37°C, 5% CO2 |
| 3 | Dye load | 20 μL | ACT:One membrane potential dye |
| 4 | Incubation | 1 h | Room temperature, dark |
| 5 | Compound (antagonist) addition | 5 μL | From 10× compound plate |
| 6 | Incubation | 1 h | Room temperature, dark |
| 7 | Agonist challenge | 5 μL | Fluid transfer performed in FLIPR |
| 8 | Read fluorescence at time=0 | T0 | Use 510–545/565–625 bandpass FLIPR filter |
| 9 | Incubation | 30 min | Room temperature in FLIPR chamber |
| 10 | Read fluorescence at time=30 | T30 | Use 510–545/565–625 bandpass FLIPR filter |
Step Notes
1. BD Biosciences black clear bottom poly-d-lysine coated 384-well plate.
4. Generate compound plate during this step.
5. Final DMSO concentration in assay plate=0.7%. Made in PBS.
6. Generate agonist plate during this step.
7. Challenge with EC90 porcine galanin (American Peptide) for antagonist mode, or CRC of porcine galanin for control, all in the presence of EC90 of forskolin (MP Biochemicals), (10 μM in 10× plate; 1 μM final concentration), and PDE inhibitor (Sigma Aldrich) (250 μM in 10× plate; 25 μM final concentration), made in PBS, final DMSO concentration ∼0.7%.
10. The T30/T0 ratio is used to graph the concentration response of test compounds.
FLIPR, fluorescent imaging plate reader; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; EC90, 90% of maximal response; CRC, concentration response curve; PDE, phosphodiesterase.