Table 1.
Example of HTS Assay Protocol Table
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Media | 40 μL | 7H9 complete media; Hyg, Km, −/+ TRIM: (control vs. test) |
| 2 | Library compounds | 1.5 μLa | 4 concentrations: 34.9 to 0.17 μM |
| 3 | Cells | 40 μL | Mycobacterium smegmatis expressing DevR–DevR |
| 4 | Incubation time | 8 h | 37°C |
| 5 | Alamar Blue | 8 μL | Redox indicator dye; 10% final solution |
| 6 | Incubation time | 36 h | 37°C |
| 7 | Assay readout | 530 nm/590 nm | Biotek microplate reader; fluorescent mode |
1. Black 384-well plate, clear bottom; 1,200 compounds per round; μFill dispenser used to load media into all wells
2. Chembridge source plates in 100% DMSO, 2 mM; 1.5 μL transfer from source plates,a 6.7 μL for dilutions
3. Prepared cells; μFill dispenser used to load cells into designated wells at 105 cells/well
4. Plates sealed to prevent evaporation
5. μFill dispenser used to load Alamar Blue
6. Plates sealed to prevent evaporation; total 30 plates (2 sets of 15); Placed on stacker, fluorescence read every 3 h
7. Biotek microplate reader; data analyzed at 24 h postaddition of Alamar Blue; compared control vs. test plates
For future screens, it is recommended that compound dilutions are performed in DMSO first, before transferring to test plates.
DMSO, dimethylsulfoxide.