Permeability assay (Caco-2 cells)
Aim: Assessment of in vitro bidirectional transport in Caco-2 cells to screen test compound for permeability and involvement of efflux transporters.
Background information: Caco-2 cells are a human colon adenocarcinoma cell line used as an in vitro model of human intestinal absorption of drugs. When cultured as a monolayer, they differentiate and polarize to form intercelular tight junctions and a brush border with efflux transporters, mimicking the human intestinal epithelium.
Transport assays are performed in both directions across the monolayer: absorption (apical to basolateral) and efflux (basolateral to apical). Test compound is added to either the apical or basolateral sides of the monolayer. Concentrations are quantified by UPLC-MS/MS using a calibration curve.
Compound requirement: 50 µl of 10 mM DMSO solution
Turnaround time: 4 weeks
- Test compound concentration: 10 µM
- 24-well plate
- Number of replicates: 2
- DMSO concentration: 1%
- Incubation time: 120 min
- Control: positive controls of efflux transporters or permeability
- Monolayer integrity marker: lucifer yellow
- Analytical method: UPLC-MS/MS with calibration curves
- Absorption and efflux permeability
- Absorption and efflux recovery
- Efflux ratio