OAT3 Inhibition – Transfected Cells
This OAT3 transporter assay is used to screen for inhibition of OAT3 by a test compound in OAT3-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 furosemide in the presence and absence of test compound in OAT3-transfected and vector control cells
- Percent inhibition by the test compound
- >50% inhibition indicates that the test compound is a significant inhibitor of OAT3
- OAT3 probe substrate furosemide at 5 μM
- Test compound at 10 μM in modified Hanks’ buffer with less than 0.8% DMSO
- OAT3-transfected HEK293 cells and vector control cells cultured in 24-well plates
- Measure uptake of furosemide in OAT3-transfected and vector control cells with and without test compound
- Single concentration of test compound
- Two cell lines: OAT3-transfected and vector control cell lines
- Four treatments as follows:
- OAT3-transfected cells with furosemide +/-test compound
- Vector control cells with furosemide +/- 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 furosemide concentration
- Determine protein concentration in cell lysate and normalize furosemide concentration to protein content
- Uptake rate of probe substrate in OAT3-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-(IROAT3-IRVC)TC / (IROAT3 – IRVC)0], where IROAT3 is the influx rate of furosemide in OAT3-transfected cells, IRVCis 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)