title: A novel fluorescent sensor for measurement of CFTR function by flow cytometry.
abstract: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting.
authors: Department of Pediatric Pulmonology, Wilhelmina Children's Hospital, University Medical Centre Utrecht, Utrecht, The Netherlands.
authors: Lodewijk A W
authors: LA
authors: Vijftigschild
authors: Cornelis K
authors: CK
authors: van der Ent
authors: Jeffrey M
authors: JM
authors: Beekman
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins; D009154:Mutation; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; C505032:CFTR protein, human; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D019833:Genistein