A novel method for measuring intracellular pH: effect of neutron irradiation on pHi of transformed cells.
A new flow cytometric method was developed for measuring the intracellular pH (pHi) of mammalian cells using a fluorescent pH indicator dye 2',7-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). Emission intensities (or their ratios) measured from BCECF-loaded cells can be converted into absolute pHi values using appropriate calibration curves. By comparison of several possible measuring and data evaluation procedures a double-ratio method was suggested as the most advantageous protocol to yield reliable intracellular pH data. This method allows pHi to be determined on a cell-by-cell basis corrected for cell volume and change in geometry of input-output optics of the flow cytometer. Our method applies a standard calibration curve and does not necessitate its reconstruction for each new set of measurements. Cells of the OKT-4 and OKT-8 hybridoma lines were exposed to neutron irradiation of different doses. Irradiated cells underwent a biphasic alkalinization; an instantaneous effect detected within 1.5 h was found to be intensified over 24 h. For the interpretation of data we suggest that the increase in cytoplasmic pH following neutron treatment is evoked by two mechanisms.
Balkay
L
L
Biomedical Cyclotron Laboratory, University Medical School of Debrecen, Hungary.
Márián
T
T
Emri
M
M
Trón
L
L
D000818:Animals; D005434:Flow Cytometry / Q000379:methods; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D009502:Neutrons; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence / Q000379:methods; D013601:T-Lymphocytes / Q000502:physiology / Q000528:radiation effects*
D005452:Fluoresceins; D005456:Fluorescent Dyes; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester