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Background:
Caco-2 cell line is a line of human epithelial colon adeno-carcinoma cells. When cultured under specific conditions they become differentiated and polarised such that morphologically and functionally resemble the epithelial cells of human small intestine. Therefore, they are considered to be a golden standard for in vitro prediction of permeability and for estimation of the potential for different efflux/uptake transporters (most importantly P-glycoprotein, P-gp) to transport the compound of interest.

Transport of compound through Caco-2 cell monolayer is used for measuring the 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. In addition, the inclusion of the P-gp inhibitor could identify wheter membrane transport is mediated by P-glycoprotein.
   Assay description
    
   Cells
      Caco-2

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

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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