title: Calcein accumulation as a fluorometric functional assay of the multidrug transporter.
abstract: Acetoxymethyl ester (AM) derivatives of various fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are actively extruded by the multidrug transporter (MDR1, P-glycoprotein-Homolya, L. et al. (1993) J. Biol. Chem. 268, 21493-21496). In the present paper we show that the measurement of the accumulation of a fluorescent cell viability marker, calcein, can be effectively used as a rapid and sensitive fluorometric and flow cytometric assay for studying P-glycoprotein function. The rate of calcein accumulation in human MDR1-expressing cells is significantly lower than in the control cells, while various drug-resistance reversing agents (verapamil, vinblastine, oligomycin, cyclosporin A and UIC2 monoclonal antibody) greatly increase calcein trapping only in the MDR1-expressing cells. Since calcein-AM is not fluorescent and free calcein is not a substrate of the multidrug transporter, the assay is readily applicable for rapid kinetic studies of the MDR1 function. Calcein has a high fluorescence intensity in the visible range, thus changes in calcein uptake can be easily visualised and MDR1-expressing and control cells separated by conventional flow cytometry.
authors.affiliation: National Institute of Haematology, Blood Transfusion and Immunology, Budapest, Hungary.
authors.forename: Z
authors.initials: Z
authors.lastname: Holló
authors.forename: L
authors.initials: L
authors.lastname: Homolya
authors.forename: C W
authors.initials: CW
authors.lastname: Davis
authors.forename: B
authors.initials: B
authors.lastname: Sarkadi
mesh_terms: D016475:3T3 Cells / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D005470:Fluorometry; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D051379:Mice; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D005452:Fluoresceins; D008562:Membrane Glycoproteins; C007740:fluorexon