| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Cells | 4 μL | 2000 cells/well |
| 2 | Incubation | 24 hrs | 37°C, 5% CO2 |
| 3 | Compound | 23 nl | in DMSO dilution |
| 4 | Incubation | 10 min | 37°C, 5% CO2 |
| 4 | Reagent | 1 μl | Carbachol (EC80) |
| 5 | Incubation | 30 min | 37°C, 5% CO2 |
| 7 | Reagents | 1 μl each | IP1-d2 and Eu-anti-IP1-Ab in cell lysis buffer, dispensing separately |
| 8 | Incubation | 30 min | Room temperature |
| 9 | Detection | HTRF mode | ViewLux plate reader |