title: Quantitative assessment of p-glycoprotein expression and function using confocal image analysis.
abstract: P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.
authors.affiliation: 1Manchester Pharmacy School,University of Manchester,Stopford Building,Manchester,M13 9PT,UK.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Hamrang
authors.affiliation: 2Faculty of Life Sciences,University of Manchester,Michael Smith Building,Manchester,M13 9PT,UK.
authors.forename: Yamini
authors.initials: Y
authors.lastname: Arthanari
authors.affiliation: 1Manchester Pharmacy School,University of Manchester,Stopford Building,Manchester,M13 9PT,UK.
authors.forename: David
authors.initials: D
authors.lastname: Clarke
authors.affiliation: 1Manchester Pharmacy School,University of Manchester,Stopford Building,Manchester,M13 9PT,UK.
authors.forename: Alain
authors.initials: A
authors.lastname: Pluen
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D000818:Animals; D004285:Dogs; D018432:Drug Resistance, Multiple; D020869:Gene Expression Profiling / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D061985:Madin Darby Canine Kidney Cells; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1