Experimental settings for optimizing incubation conditions of an automated UGT phenotyping assay using a panel of 10 UGT probe substrates in HLMs
| Experimental Condition | Value |
|---|---|
| Material | Pooled HLMs (150 donors) |
| 15 HLMs from single donors | |
| Enzyme concentration | 1 mg/ml |
| Incubation volume | 0.4 ml |
| Alamethicin treatment | 10 µg/ml, for 30 min at 4°C |
| Preincubation step | 5 min, shaking at 37°C |
| Incubation time | 0.5–30 min, shaking at 37°C |
| Incubation buffer | Potassium phosphate (100 mM at pH 7.4) |
| Tris-HCl (100 mM at pH 7.4) | |
| MgCl2 | 0, 1, 2, 5, 8, and 10 mM |
| UDPGA | 1, 2.5, 3.75, 5, 8, 12, 16, and 25 mM |
| BSA | 0%, 0.1%, 0.2%, 0.5%, 1.0%, 1.5%, and 2% w/v |