Table 1.
Workflow of the Optimized Epidermal Growth Factor Receptor Biosensor Assay
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Library compounds | 5 μL | 100 μM in 10% DMSO (v/v) |
| 2 | Low control | 5 μL | 100 μM gefitinib in 10% DMSO (v/v) |
| 3 | High control | 5 μL | 10% DMSO (v/v) |
| 4 | Cell plating | 45 μL | 5000 A549-EGFRB cells in cell culture RPMI 1640 media |
| 5 | Incubation time | 16 h | 37°C, 5% CO2 |
| 6 | Incubation time | 70 min | 500 nM EGF stimulation in cell culture RPMI 1640 media |
| 7 | Fix | 50 μL | 4% PFA for 20 min |
| 8 | Wash | 50 μL | Two times with 1× PBS |
| 9 | Nuclear staining | 50 μL | 10 μM Hoechst in 1× PBS for 10 min |
| 10 | Wash | 50 μL | Two times with 1× PBS |
| 11 | Assay readout | 350 nm/455 nm and 490 nm/525 nm (ex/em) | 20× objective imaging using IN Cell Analyzer 2000 microscope system |
| 12 | Image analysis | — | Multiparametric analysis using Developer Toolbox 1.7 software |
1–3. Dispensing with the PP-384-M Personal Pipettor using a custom 384 head.
4. Cells diluted in media and dispensed into 384-well assay plate with Multidrop 384.
5,6. 384-well assay plates stored in the Cytomat automated temperature-controlled incubator.
6–10. Aspirating with the ELx405 washer and dispensing with the Multidrop 384.
11. Readout performed on an automated platform; 19 s per well with total imaging time of 120 min per 384-well microtiter plate.
DMSO, dimethylsulfoxide; EGF, epidermal growth factor; PFA; PBS, phosphate-buffered saline; ex/em, excitation and emission.