ExpressPlus Bidirectional Permeability Through Caco-2 Cell Monolayers
This assay is used to determine the permeability of a test compound through Caco-2 cell monolayers in the apical-to-basolateral and basolateral- to-apical direction
Catalog number EA202
Required from Customer
- Either a minimum of 300 µL of test compound at 10 mM in DMSO, or 5 mg of powder
- Exact molecular mass of the test compound and its salt form
- MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.
- The percent recovery of the test compound from the Transwell® wells containing Caco-2 cell monolayers
- The apparent permeability (Papp) in both directions
- The absorption potential of a test compound classified as either Low or High using the following criteria for the apical-to-basolateral (A to B) direction:
- Low: Papp <1.0 x 10-6 cm/s
- High: Papp ≥1.0 x 10-6 cm/s
- The efflux ratio (Papp B to A)/(Papp A to B)
- The efflux is classified as Significant when:
- efflux ≥3.0 and Papp (B to A) ≥ 1.0 x 10-6 cm/s
- Test compound at 5 µM in HBSSg with maximum DMSO concentration less than 1%
- Confluent monolayers of Caco-2 cells, 21 to 28 days old
- Receiver well with 1% BSA in modified Hanks buffer HBSSg
- Apical and basolateral side at pH 7.4 ± 0.2
- Two monolayers dosed in each direction (N=2)
- Dose apical side for (A to B) assessment
- Dose basolateral side for (B to A) assessment
- Sample both apical and basolateral sides at 120 minutes
- Determine the concentrations of test compound using a generic LC-MS/MS method with a minimum 4 point calibration curve
- The quality of the monolayer batch is verified using control compounds before the monolayers are released for use
- The quality of each monolayer used in the assay is verified by a TEER measurement and by calculating the Papp for a control compound
- The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
- This assay broadly classifies the Papp of a test compound—see EA212 for an alternative.
- For reproducible results the solubility of the test compound must be greater than 5 µM in HBSSg with 1% DMSO.