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EA272

OAT3 IC50 Determination – Transfected Cells

This assay is used to determine the IC50 of a test compound for inhibition of OAT3 in OAT3-transfected HEK293 cells.

Required from Customer

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

Deliverables

  • Normalized uptake of furosemide in the presence and absence of test compound in OAT3-transfected and vector control cells
  • IC50 of the test compound

Substrate

  • OAT3 probe substrate PAH at 10 μM
  • Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

  • OAT3-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

  • Measure uptake of PAH in OAT3-transfected and vector control cells with and without test compound
  • 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 probenecid
    • Duplicate incubations (n=2)
  • Two cell lines: OAT3-transfected and vector control cell lines
  • Treatments performed in each cell line:
    • Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
    • Buffer is aspirated and replaced with dosing solution
    • Incubate cells for 5 min at 37°C
    • Terminate the incubation and lyse the cells
    • Analyze cell lysate using LC-MS/MS to determine PAH concentration
    • Determine protein concentration in cell lysate and normalize PAH concentration to protein content

Assay QC

  • Uptake rate of probe substrate in OAT3-transfected vs. vector control cells
  • Inhibition of probe substrate uptake by a known inhibitor of OAT3

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 Hanks’ buffer containing < 0.8% DMSO 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.