title: Sensitivity of chloride efflux vs. transepithelial measurements in mixed CF and normal airway epithelial cell populations.
abstract: OBJECTIVE
  While the Cl(-) efflux assays are relatively straightforward, their ability to assess the efficacy of phenotypic correction in cystic fibrosis (CF) tissue or cells may be limited. Accurate assessment of therapeutic efficacy, i.e., correlating wild type CF transmembrane conductance regulator (CFTR) levels with phenotypic correction in tissue or individual cells, requires a sensitive assay.
  
  
  METHODS
  Radioactive chloride ((36)Cl) efflux was compared to Ussing chamber analysis for measuring cAMP-dependent Cl(-) transport in mixtures of human normal (16HBE14o-) and cystic fibrosis (CF) (CFTE29o- or CFBE41o-, respectively) airway epithelial cells. Cell mixtures with decreasing amounts of 16HBE14o- cells were evaluated.
  
  
  RESULTS
  Efflux and Ussing chamber studies on mixed populations of normal and CF airway epithelial cells showed that, as the number of CF cells within the population was progressively increased, the cAMP-dependent Cl(-) decreased. The (36)Cl efflux assay was effective for measuring Cl(-) transport when ≥ 25% of the cells were normal. If < 25% of the cells were phenotypically wild-type (wt), the (36)Cl efflux assay was no longer reliable. Polarized CFBE41o- cells, also homozygous for the ΔF508 mutation, were used in the Ussing chamber studies. Ussing analysis detected cAMP-dependent Cl(-) currents in mixtures with ≥1% wild-type cells indicating that Ussing analysis is more sensitive than (36)Cl efflux analysis for detection of functional CFTR.
  
  
  CONCLUSIONS
  Assessment of CFTR function by Ussing analysis is more sensitive than (36)Cl efflux analysis. Ussing analysis indicates that cell mixtures containing 10% 16HBE14o- cells showed 40-50% of normal cAMP-dependent Cl(-) transport that drops off exponentially between 10-1% wild-type cells.
authors.affiliation: Children's Hospital Oakland Research Institute, Oakland, USA.
authors.forename: Beate
authors.initials: B
authors.lastname: Illek
authors.forename: Dachuan
authors.initials: D
authors.lastname: Lei
authors.forename: Horst
authors.initials: H
authors.lastname: Fischer
authors.forename: Dieter C
authors.initials: DC
authors.lastname: Gruenert
mesh_terms: D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport; D010641:Phenotype; D011868:Radioisotopes / Q000737:chemistry; D020545:Respiratory Mucosa / Q000378:metabolism*
chemical_list: D002712:Chlorides; D011868:Radioisotopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP