Measurement of halide efflux from cultured and primary airway epithelial cells using fluorescence indicators.
The use of the halide-sensitive fluorescent probes (6-methoxy-N-(-sulphopropyl)quinolinium (SPQ) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)) to measure chloride transport in cells has now been established as an alternative to the halide-selective electrode technique, radioisotope efflux assays and patch-clamp electrophysiology. We report here procedures for the assessment of halide efflux, using SPQ/MQAE halide-sensitive fluorescent indicators, from both adherent cultured epithelial cells and freshly obtained primary human airway epithelial cells. The procedure describes the calculation of efflux rate constants using experimentally derived SPQ/MQAE fluorescence intensities and empirically derived Stern-Volmer calibration constants. These fluorescence methods permit the quantitative analysis of CFTR function.
Department of Gene Therapy, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London SW3 GLR, UK.
Felix
F
Munkonge
Eric W F W
EW
Alton
Charlotte
C
Andersson
Heather
H
Davidson
Anca
A
Dragomir
Aleksander
A
Edelman
Ray
R
Farley
Lena
L
Hjelte
Gerry
G
McLachlan
Myra
M
Stern
Godfried M
GM
Roomans
D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology; D005456:Fluorescent Dyes; D006801:Humans; D007454:Iodides / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D011806:Quinolinium Compounds; D020545:Respiratory Mucosa / Q000187:drug effects / Q000502:physiology*
C505032:CFTR protein, human; D002712:Chlorides; D005456:Fluorescent Dyes; D007454:Iodides; D011806:Quinolinium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C085493:N-(ethoxycarbonylmethyl)-6-methoxyquinolinium; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium