title: A simplified approach to determining P-glycoprotein expression in peripheral blood mononuclear cell subsets.
abstract: P-glycoprotein (P-gp), encoded by the MDR-1 (multidrug resistance) gene mediates the cellular efflux of several therapeutic agents with the potential of treatment failure. The differential expression of P-gp in many localised tissues and cells of the hematopoietic system implies diverse physiological and pharmacological roles. The exact function of P-gp involved in multidrug resistance remains unclear owing to the numerous discrepancies between different laboratories. The ability to characterise accurately P-gp expression has important clinical implications. However, a complete consensus recommendation regarding methods of P-gp detection has been difficult to reach. With the advancement in immune technology and new commercially available antibodies, we describe a simplified direct immunofluorescent assay capable of detecting surface P-gp expression in peripheral blood mononuclear cells (PBMCs) and subpopulations of lymphocytes in vivo by dual colour flow cytometry. Results were expressed as mean increase in fluorescence (MI) compared to isotypically matched controls. Using this assay, differential basal P-gp expression was found to exist in the following significant hierarchy CD56+ (MI=0.684+/-0.273; n=15)>CD8+ (MI=0.312+/-0.117; n=15)>CD4+ (MI=0.194+/-0.086; n=15). This method is rapid and reproducible and has potential use for in vitro and in vivo application.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Liverpool, 70 Pembroke Place, Block H, First Floor, L69 3GF, Liverpool, UK. jford@liv.ac.uk
authors.forename: J
authors.initials: J
authors.lastname: Ford
authors.forename: P G
authors.initials: PG
authors.lastname: Hoggard
authors.forename: A
authors.initials: A
authors.lastname: Owen
authors.forename: S H
authors.initials: SH
authors.lastname: Khoo
authors.forename: D J
authors.initials: DJ
authors.lastname: Back
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000276:immunology / Q000378:metabolism*; D000328:Adult; D000911:Antibodies, Monoclonal / Q000276:immunology; D015704:CD4 Antigens / Q000032:analysis / Q000276:immunology; D019002:CD56 Antigen / Q000032:analysis / Q000276:immunology; D016827:CD8 Antigens / Q000032:analysis / Q000276:immunology; D003116:Color; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D010799:Phycoerythrin; D015203:Reproducibility of Results; D016176:T-Lymphocyte Subsets / Q000378:metabolism*; D013997:Time Factors
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D019002:CD56 Antigen; D016827:CD8 Antigens; D005456:Fluorescent Dyes; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate