EA268

P-gp IC50 Determination – Caco-2

This P-gp transporter assay is used to determine the IC50 of a test compound for inhibition of P-gp in Caco-2 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
  • Solubility and chemical stability of the test compound in Hanks’ buffer
  • MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.

Deliverables

  • Apparent permeability (Papp) of digoxin in the basolateral-to-apical direction, in the presence and absence of test compound
  • IC50 of the test compound

Substrate

  • P-gp probe substrate digoxin 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

  • Confluent monolayers of Caco-2 cells, 21-28 days old, in Transwell® dual-chamber plates, with apical and basolateral pH 7.4

Assay Conditions

  • Monolayers pretreated and co-dosed with test compound on both apical and basolateral sides
  • 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 Valspodar
    • Duplicate incubations (n=2)
  • Dose digoxin on basolateral side only for B to A permeability
  • Sample both apical and basolateral sides at 120 minutes
  • Determine the concentration of digoxin using a generic LC-MS/MS method with a minimum 4-point calibration curve

Assay QC

  • Verify the quality of the cell monolayer batch using control compounds before the monolayers are released for use
  • Verify the quality of each monolayer used in an assay by a pre-experiment TEER measurement and by calculating the Papp for the co-dosed control compound, Lucifer yellow

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.

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