| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Cells | 3 μl | 2000 cells/well |
| 2 | Incubation | 16-20 hrs | 37°C, 5% CO2 |
| 3 | Reagent | 3 μl | No-wash calcium dye |
| 4 | Incubation | 60 min | 37°C, 5% CO2 |
| 5 | Compound | 23 nl | in DMSO dilution |
| 6 | Incubation | 10 min | Room temperature |
| 7 | Detection | Basal signal | 10 reads at 1 Hz |
| 8 | Reagent | 2 μl | Carbachol (EC80) |
| 9 | Detection | Antagonist response | 4-minute reading at 1Hz |