Required from Customer

• Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 10 mg of powder
• Exact molecular mass of test compound and its salt form
• Solubility of test compound in Hanks’ buffer
• MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

• Normalized uptake of ³H-taurocholic acid in the presence and absence of test compound in hBSEP-expressing vesicles
• IC₅₀ of the test compound

Substrate

• BSEP probe substrate ³H-taurocholic acid at 1 μM
• Test compound at six concentrations (serial diluations from 100 µM) in Hepes Influx Buffer

Assay System

• hBSEP-expressing vesicles in the presence of ATP vs. AMP

Assay Conditions

• Measure uptake of ³H-taurocholic acid in hBSEP-expressing vesicles with and without test compound, in the presence of ATP vs. AMP
• Six concentrations of test compound
  • Single incubation (n=1) per concentration
• Negative control: vehicle only
  • Duplicate incubations (n=2)
• Positive control: single concentration of known inhibitor CsA
  • Duplicate incubations (n=2)
• Treatments performed in in the presence of ATP and AMP (separately):
  • Incubate cells for 30 min at 37°C
  • Terminate the incubation
  • Measure radioactivity of processed samples to determine ³H-taurocholic acid concentration

Assay QC

• Uptake rate of probe substrate in hBSEP-expressing vesicles in the presence of ATP vs. AMP
• Inhibition of probe substrate uptake by a known inhibitor of BSEP

Notes

1. The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
2. The solubility of the test compound in Hepes Influx Buffer must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.