OATP1B1 Inhibition – Transfected Cells
This assay is used to screen for inhibition of OATP1B1 by a test compound in OATP1B1-transfected HEK293 cells at a single concentration
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
- Relevant solubility of the test compound
- MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.
- Normalized uptake of atorvastatin in the presence and absence of test compound in OATP1B1-transfected and vector control cells
- Percent inhibition by the test compound
- >50% inhibition indicates that the test compound is a significant inhibitor of OATP1B1
- OATP1B1 probe substrate Atorvastatin at 0.15 μM
- Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO
- OATP1B1-transfected HEK293 cells and vector control cells cultured in 24-well plates
- Measure uptake of atorvastatin in OATP1B1-transfected and vector control cells with and without test compound
- Single concentration of test compound
- Two cell lines: OATP1B1-transfected and vector control cell lines
- Four treatments as follows:
- OATP1B1-transfected cells with atorvastatin +/-test compound
- Vector control cells with atorvastatin +/- test compound
- Perform assay in duplicate (N=2 per treatment)
- Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
- Buffer is aspirated and replaced with test compound dosing solution
- Incubate cells for 5 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine atorvastatin concentration
- Determine protein concentration in cell lysate and normalize atorvastatin concentration to protein content
- Uptake rate of probe substrate in OATP1B1-transfected vs. vector control cells
- The results from this assay are provided to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
- The solubility of the test compound in Hanks’ buffer containing < 0.8% DMSO must be greater than the test concentration. If the solubility of the test compound is unavailable, Absorption Systems can conduct a solubility assessment at an additional charge.
- Percent inhibition is calculated as 100 x [1-(IR1B1-IRVC)TC / (IR1B1 – IRVC)0], where IR1B1 is the influx rate of atorvastatin in OATP1B1-transfected cells, IRVC is the influx rate in vector control cells, “TC” means in the presence of test compound, and “0” means in the absence of test compound.
- The customer can request:
- that a positive control be performed in parallel (additional fees apply)