EA290 – MATE2K IC₅₀ MATE2K-HEK293

ExpressPlus MATE2K IC50 Determination Using Transfected Cells

This assay is used to determine the IC₅₀ of a test compound for inhibition of MATE2K in MATE2K-transfected HEK293 cells

Required from Customer

• Either a minimum of 300 µL of test compound at 20 mM in DMSO, or 10 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.

Deliverables

• Normalized uptake of Metformin in the presence and absence of test compound in MATE2K-transfected and vector control cells
• IC₅₀ of the test compound

Substrate

• MATE2K probe substrate Metformin at 50 μM
• Test compound at six concentrations (serial diluations from 100 µM) in modified Hanks’ buffer with less than 0.8% DMSO

Assay System

• MATE2K-transfected HEK293 cells and vector control cells cultured in 24-well plates

Assay Conditions

• Measure uptake of Metformin in MATE2K-transfected and vector control cells with and without test compound
• Six concentrations of test compound
  • Single incubation (n=1) per concentration
• Negative control: vehicle only
  • Duplicate incubations (n=2)
• Positive control: single concentration of known inhibitor Pyrimethamine
  • Duplicate incubations (n=2)
• Two cell lines: MATE2K-transfected and vector control cell lines
• Treatments performed in each cell line:
  • Wells are equilibrated with blank buffer containing 1% BSA prior to initiating the assay
  • Buffer is aspirated and replaced with 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 Metformin concentration
  • Determine protein concentration in cell lysate and normalize Metformin concentration to protein content

Assay QC

• Uptake rate of probe substrate in MATE2K-transfected vs. vector control cells
• Inhibition of probe substrate uptake by a known inhibitor of MATE2K

Notes

1. 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.
2. 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.