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. Author manuscript; available in PMC: 2013 Mar 1.
Published in final edited form as: J Pharmacol Toxicol Methods. 2012 Feb 26;65(2):64–74. doi: 10.1016/j.vascn.2012.02.002

Fig. 1.

Fig. 1

Fl-t-pgp principles of operation. Fl-t-pgp consists of purified Pgp reconstituted into the bilayers of unilamellar liposomes that contain drug-binding fluorescent sensor molecules in their aqueous interior. Prior to the actual assay, the Pgp substrate S-HR is introduced into a preparation of Fl-t-pgp and passively diffuses through the membrane to reach a state of equilibrium. Upon entering the aqueous interior of the Fl-t-pgp particle, the substrate rapidly binds to the fluorescent sensor, resulting in a new and stable fluorescence baseline (not shown). 1. To conduct a transport assay, “Fl-t-pgp reagent” in the wells of a microplate is placed in a fluorescence plate reader, and measurement is initiated. 2. ATP is injected. 3. Substrate is actively pumped by Pgp against a concentration gradient into the interior of the Fl-t-pgp particle, and the influxed substrate is rapidly bound by the encapsulated fluorescent sensor. 4. A linear, time-dependent alteration of the fluorescence intensity is observed.