This assay uses inside-out vesicles to assess whether the test compound is a potential substrate or inhibitor of the efflux transporter BSEP
Catalog numbers: EA235
Required from Customer
- 50 mg of test compound powder
- Molecular weight of the test compound and its salt form
- Relevant solubility of test compound
- The MSDS and handling and storage information, e.g., light sensitive, store at -20ºC, etc.
Deliverables
- Determination of substrate classification: positive when the ratio of the influx rates in BSEP vesicles + ATP vs. BSEP vesicles + AMP >2
- IC50 of the test compound
Substrate
Study I:
- Test compound at three concentrations in Hepes Influx Buffer (10mM Hepes-Tris, pH 7.4) with ≤ 0.8% DMSO
Study II:
- BSEP probe substrate, 3H-taurocholic acid
- Test compound at seven concentrations in modified Hanks’ buffer with <0.8% DMSO
Assay System
BSEP inside-out vesicles in the presence of ATP or AMP
Assay Conditions
Pre-study work is done to establish the calibration range and method reproducibility, and to evaluate elution optimization, chemical stability, solubility and non-specific binding
Study I: BSEP substrate assessment
- Measure uptake of test compound in BSEP inside-out vesicle
- Three concentrations of test compound
- Two conditions:
- Incubated with ATP
- Incubated with AMP
- Perform assay in triplicate (N=3 per treatment)
- Incubate cells for 5, 15 and 30 min at 37°C
- Analyze dosing solution and filter plate eluant samples using LC-MS/MS to determine test compound concentration
Study II: BSEP inhibitor assessment
- Measure uptake of 3H-TCA in BSEP inside-out vesicles with and without test compound
- Test compound at six concentrations
- Negative control: vehicle only
- Positive control: CsA
- Eight treatments per condition as follows:
- BSEP vesicles with 3H-TCA +/- test compound (6 concentrations) or positive control incubated with ATP
- BSEP vesicles with 3H-TCA +/- test compound (6 concentrations) or positive control incubated with AMP
- Perform assay in duplicate (N=2 per treatment)
- Incubate cells for 30 min at 37oC
- Analyze filter plate eluant samples using scintillation counting to determine 3H-TCA concentration
Options
- The customer may be asked to specify:
- The concentrations of the test compound
- Substrate assessment can be performed using radio-labeled test compound