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Permeability- MDCK Wild Type

Background:
MDCK cells (wild type) are Madin-Darby canine kidney epithelial cells. They endogenously express different transporters, such as canine P-glycoprotein (P-gp). Therefore, it is recommended to analyse test compound on both MDCK and MDCKII-MDR1 cell monolayer and determine the real role of human P-gp on transport of test compound.
 
Transport of compound through MDCK cell monolayer is used for measuring rate of membrane transport, expressed as apparent (Papp) permeability. Resulting Papp values from both transport directions (apical to basolateral side, and basolateral to apical side) are used for calculation of efflux ratio, as well as of net flux ratio (if MDCKII-MDR1 data are available).
   Assay description

   Cells
      MDCK wild type (wt)

   Direction
      apical to basolateral  (A2B)
      basolateral to apical (B2A)
      with or without P-gp inhibitor (elacridar)

   Compound concentration
      10µM (1% DMSO)

   Compound requirements
      50µl of 10mM stock solution or
      1-2 mg of dry matter

   Incubation details
      medium: Dulbecco’s PBS (pH 7.4)
      calibration curve: optional

   Detection method
      LC-MS/MS with internal standard

   Results
      Papp(A2B),  Papp(B2A)
      efflux ratio, recovery, net flux ratio

  Assay controls
     reference compounds:  amprenavir and propranolol 
     cell monolayer integrity control: lucifer yellow

Assay details adjustable to client’s and/or project specific requests

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