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
Deliverables
- Normalized uptake of test compound at each concentration in OATP1B1-transfected and vector control cells
- Determination of substrate classification: positive when the ratio of the influx rates in OATP1B1-transfected cells vs. vector control cells >2.0
Substrate
- Test compound at 0.5 and 5 µM in HBSSg (pH 7.4) with maximum DMSO concentration not greater than 1%
Assay System
- OATP1B1-transfected HEK293 cells and vector control cells cultured in 24-well plates
Assay Conditions
- Measure uptake of test article in-transfected and vector control cells
- Two concentrations of test article and two sampling time points
- Treatments per transporter as follows (repeated for each concentration and incubation duration):
- Transfected cells with test article
- Vector control cells with test article
- 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 article dosing solution
- Incubate cells for 2 and 10 min at 37°C
- Terminate the incubation and lyse the cells
- Analyze cell lysate using LC-MS/MS to determine test article concentration
- Determine protein concentration in cell lysate and normalize probe substrate concentration to protein content
Batch QC
- The quality of the cell batch is verified before the cells are used (uptake rate of a probe substrate in OATP1B1-transfected vs. vector control cells)
Options
The customer can request:
- An in-study positive control for an additional fee
Notes
- The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data.
