Lab Services - Contract Services


Print this document by click/touch on the red printer icon below.

Transporter Assay Requirement

Transporter Assay with stable transfected HEK293 cells

Stable transfected HEK293 cells expressing transporter protein (OATPs, OCTs, OATs, NTCP and ASBT) can be used to analyze whether the test compound is a substrate or potential substrate of these transporters.

1. Customer Requirements

Either a minimum of 100 µL of test compound at 100 mM, or 5 mg of powder (for one transporter assay).
Exact molecular mass of test compound and its solubility MSDS or handling and storage information.

2. Assay system

Well characterized stable transfected HEK293 cells expressing uptake transporter and vector
control cells cultured in 24-well plates

3. Assay Conditions

3.1 Uptake assay (when the customer is able to analyze the test compound)
• Measure uptake of test substance in transporter transfected and vector control cells
• Two concentrations of test article and two sampling time points
• Perform assay in duplicate (N=2 per treatment)
• Incubate cells for two time points at 37 °C
• Terminate the incubation
• The plate will be frozen (dry) at -20 °C and send to the customer on dry ice

3.2 Competition assay (to test if the test compound is a potential transporter substrate)
• Measure uptake of reference substrate with and without test substance in transfected and vector control cells
• Two concentrations of test article and two sampling time points
• Perform assay in duplicate (N=2 per treatment)
• Incubate cells for two time points at 37 °C
• Terminate the incubation and lyse the cells
• Analyze cell lysate by fluorometric assay or scintillation counting to determine the uptake of reference substrate
• Determine protein concentration in cell lysate and normalize the uptake of reference substrate concentration to protein content

3.3 Positive control
For both assays an additional positive control with reference substrate and inhibitor will be performed to demonstrate the uptake function of transporters.

4. Results (only for competition assay)

  • Normalized inhibition effect of test compound at each concentration
  • The results from this assay are sent to the customer in the PDF format, which may include graphical presentations of data.
Q