Table 1 Assay optimization parameters.
Parameter | Conditions tested* | Duration of drug treatment | Optimal conditions |
|---|---|---|---|
Evaporation in 96-well microplates | |||
Drugs stored in flat-bottom culture microplates | 4 °C, −20 °C | 48 h | Short-term storage (<7 days) at 4 °C or −20 °C |
Drugs stored in different 96-well microplates | PCR, flat-bottom culture plates; −20 °C | 48 h, 72 h | PCR plates with aluminum sealing tape |
Cells incubated in flat-bottom culture microplates in the incubator | Bortezomib, DMSO | 24 h, 48 h, 72 h | Avoid perimeter wells (rows A and H; columns 1 and 12) due to edge effects |
Edge effect in the incubator | Bortezomib, DMSO, PBS | 24 h | Avoid perimeter wells (rows A and H; columns 1 and 12) due to edge effects |
Cell density and drug sensitivity | |||
Seeding density | 5.0 × 103, 7.5 × 103 or 1.0 × 104 cells per well | 24 h | 7.5 × 103 cells per 96-well |
Sensitivity to drug solvent (DMSO) | 0.33, 0.5, 1, 2, 5, 10, 20, 30% (v/v) DMSO | 24 h | <1% (v/v) DMSO |
Medium | |||
Medium type | Growth medium + 0% FBS, 5% FBS, 10% FBS, 15% FBS or HuMEC serum-free medium | 24 h | Growth medium + 10% FBS |
Medium volume | 100 µl, 200 µl, 240 µl growth medium + 10% FBS | 24 h | 100 µl |
Medium/drug renewal | With, without medium/drug renewal every 24 h | 24 h, 48 h, 72 h | Without medium/drug renewal |
Antibiotics | With, without penicillin-streptomycin | 24 h | With or without penicillin-streptomycin |
Controls | |||
DMSO controls | Matched DMSO concentration controls, single DMSO control | 24 h | Matched DMSO concentration controls |
Resazurin | |||
Mode of resorufin detection | Absorbance, fluorescence | 24 h | Absorbance or fluorescence |
Incubation time | 1 h, 2 h, 4 h, 6 h | 24 h | ≥4 h |
Concentration | 5%, 10%, 15%, 20% resazurin | 24 h | 10% resazurin |
Cross-reactivity with pharmaceutical compounds | Growth medium + 10% FBS (without cells) incubated with bortezomib, carboplatin, or DMSO | 24 h | No cross-reactivity observed |
