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