title: Measurement of intracellular pH in cultured cells by flow cytometry with BCECF-AM.
abstract: This study evaluates the suitability of flow cytometry with the fluorochrome BCECF for measuring the intracellular pH (pHi) of cultured cells, and monitors the changes in pHi in murine hybridoma in batch culture and chick embryo fibroblast in monolayer culture (5th passage). The technique produced highly reproducible, repeatable results. The theoretical sensitivity from the calibration curve was 0.0004 pH units. But analysis of the standard deviation of the histogram of the green/red fluorescence ratios indicated a mean sensitivity of 0.08 (0.07-0.09) pH units. Interference due to cell size, fluorochrome incorporation and esterases were minimized by establishing a calibration curve with the cells whose pHi was to be measured using the 525/610 nm fluorescence ratio after excitation at 488 nm. The pHi of exponentially growing, batch cultured hybridomas was 7.50 at the start of culture. pHi increased during the exponential growth phase and dropped towards cell death. The pHi of the chick fibroblasts in monolayer culture was 7.30.
authors.affiliation: Laboratoire de Biochimie Médicale l. Faculté de Médecine de Nancy, Vandoeuvre-les-Nancy, France.
authors.forename: P
authors.initials: P
authors.lastname: Franck
authors.forename: N
authors.initials: N
authors.lastname: Petitipain
authors.forename: M
authors.initials: M
authors.lastname: Cherlet
authors.forename: M
authors.initials: M
authors.lastname: Dardennes
authors.forename: F
authors.initials: F
authors.lastname: Maachi
authors.forename: B
authors.initials: B
authors.lastname: Schutz
authors.forename: L
authors.initials: L
authors.lastname: Poisson
authors.forename: P
authors.initials: P
authors.lastname: Nabet
mesh_terms: D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006825:Hybridomas / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester