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.
Munkonge
Felix
F
Department of Gene Therapy, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London SW3 GLR, UK.
Alton
Eric W F W
EW
Andersson
Charlotte
C
Davidson
Heather
H
Dragomir
Anca
A
Edelman
Aleksander
A
Farley
Ray
R
Hjelte
Lena
L
McLachlan
Gerry
G
Stern
Myra
M
Roomans
Godfried M
GM
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