BCRP Substrate – Caco-2 Cells (+/- Inhibitor)
This assay is used to determine the interaction of a test compound with BCRP using Caco-2 cell monolayers with and without a BCRP inhibitor
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 test compound and its salt form
- Solubility of test compound in Hanks’ buffer
- MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.
- The percent recovery of the test compound from Transwell® wells containing cell monolayers
- The apparent permeability (Papp) in both directions with and without inhibitor
- The efflux ratio (Papp B to A)/(Papp A to B) with and without inhibitor
- BCRP substrate classification:
- Positive when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio ≥2.0
- Negative when efflux ratio ≥3.0 in the absence of inhibitor and relative efflux ratio <2.0, or when efflux ratio <3.0 in the absence and presence of inhibitor
- Requires further study when the effux ratio ≥3.0 in the absence and presence of inhibitor
- Test compound at 5 µM in HBSSg with maximum DMSO concentration not greater 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
- Assay run in duplicate (N=2)
- Single test compound concentration at 5 µM
- Dose apical side for A to B assessment and basolateral side for B to A assessment
- Sample both donor and receiver sides at 120 minutes
- Determine the concentrations of test compound using a generic LC-MS/MS method with a minimum 6-point calibration curve
- The quality of the cell monolayer batch is verified using control compounds before the monolayers are used
- 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.
- Monolayer integrity will be verified by a pre-experimental TEER.
- This screening assay does not provide definitive information about the BCRP compound interactions or the apparent Km.
- Assay conditions with the inhibitor include:
- A 30 minute pre-incubation with Ko143
- Assay conditions without the inhibitor include:
- A 30 minute pre-incubation with buffer