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it would be of great help if you can tell me about internal standards
used in Caco-2 permeability assay. i want to know whether internal
standard in Caco-2 permeability assay is added in the same well
containing drug whose permeability is to be determined. But this would
be same as cocktail approach. so during permability assay, should we
add drug and internal standard in separate well (external standard)?
Is it required to perform viability assay frequently in caco-2?
Because we come to know it using culturing Caco-2 cells on medium?
waiting for your kind reply
Sachin Ramrao Patil
Deptt of Pharm.Tech Formulations
NIPER, SAS Nagar
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The following message was posted to: PharmPK
It is of common procedure to co-incubate with your test compound a
molecule of low permeability (and not subject to active (enzyme-
The apparent permability of that compound should remain low with a
functional Caco-2 monolayer, any increase demonstrating
deteriorations. Therefore, determination of its Papp combined to
measurement of TEER (transepithelial electric resistance) before/after
transport allows to validate each experimental well (integrity of cell
monolayer). Such low Papp molecule is often designated as an internal
Two widely used examples are radiolabelled mannitol (14C) and lucifer
yellow. The former requires radiodetection and all radioactivity-
related constraints. The latter is detected in fluorescence. Other
possibilities may exist, including IS monitored in LC-MS/MS. It should
be checked that IS detection is reliable in the tested experimental
With external standard, it is much more difficult to demonstrate that
the experimental conditions you used in apical side (pH, formulation,
test compound itself) left the cell monolayer and tight-junctions
Hope that helps.
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