Patent ID: 7122329

Claim:
A method for determining the activity and/or quantity of a transport protein of interest being localized either to the basolateral or to the apical plasma membrane of polarized cells comprising the steps of: (a) providing a well sealed confluent culture of polarized cells supposed to express said transport protein of interest grown on a permeable support, ensuring the formation of tight junctions among said cells of said culture, forming thereby separate basolateral and apical compartments on the opposite sides of said confluent cell culture; (b) adding a cell permeable non-fluorescent derivative of a fluorescent compound into one of the compartments, which is opposite to the plasma membrane of said polarized cells where the transport protein of interest is localized, said derivative being cleavable by intracellular enzymes to form a fluorescent compound that is a substrate of said transport protein of interest; (c) incubating the cells in the presence of said non-fluorescent derivative; (d) measuring the fluorescence intensity of a sample taken from the compartment opposite to the one where said non-fluorescent derivative was added, said intensity being indicative of the presence and activity of said transport protein of interest; and optionally (e) calculating the measure of activity and/or quantity of said transport protein of interest being present in said cells on the basis of the fluorescence intensity value measured in step (d) and on calibration curves and/or empirical correlations established previously.