Fluorosome®trans-pgp Inhibition Assay Procedure and Data

  1. Aliquot Fluorosome®trans-pgp, together with probe substrate, into the wells of a 96 half-well plate or a 384 well plate.
  2. Add the desired concentration of test drug as a DMSO stock solution to appropriate wells.
  3. Run the plate in an injecting fluorescence plate-reading spectrophotometer. For each well, the spectrophotometer is programmed to:
    • Read the sample for 20 sec to establish a baseline – no Pgp transporter activity.
    • Inject ATP into the sample – activate the Pgp transporter.
    • After 6 sec equilibration, follow the fluorescence change for 34 sec – Pgp transporter activity (see Figure 1 for a sample run).
    • At run completion (60 sec/well), the software immediately determines the slope of the fluorescence change with time (Pgp transporter activity).  Percent inhibition is the ratio of the slope obtained for the well with test drug relative to that for a control well containing only DMSO (no test drug).
  4. Repeat at different concentrations of test drug, measuring the percent inhibition for each.  Fitting of the inhibition data is used to estimate IC50 (see Figure 2 for plots for two drugs known to interact with Pgp).

Raw data from fluorescence plate reader

tfcbio-trans-pgp assay

Figure 1. Raw output of the inhibition of Pgp transport by cyclosporin A at 20 and 2.5 µM relative to control. The instrument used for this assay was the BMG LABTECH NOVOstar fluorescence plate reading spectrophotometer.

Calculation of IC50 values for representative Pgp inhibitors

assay plots

Figure 2. Fluorosome®trans-pgp 10 minute, 8 point IC50 determinations for representative Pgp inhibitors.