Application of a new pH-sensitive fluoroprobe (carboxy-SNARF-1) for intracellular pH measurement in small, isolated cells

Abstract

We report the use of a new pH-sensitive dualemission fluoroprobe, carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1) for ratiometric recording of intracellular pH (pH_i) in small isolated cells. The method is illustrated with pH_i measurement in single type-1 cells (cell diameter ∼10 μm) isolated from the carotid body of the neonatal rat. Carboxy-SNARF-1 is loaded using bath application of the acetoxymethyl ester. When excited at 540 nm, the fluoroprobe gives strong, inversely related emission signals at 590 nm and 640 nm. Stable ratiometric recordings of pH_i can be achieved from a single cell (pH_i 8.5-6.5) for up to 50 min. Photobleaching of the probe is minimised by illuminating at relatively low light intensity (50 W xenon lamp with 0.2% transmission neutral density filter). The probe can be calibrated in situ using the nigericin technique and this is in good quantitative agreement with the independent null-point technique (extracellular weak acid/weak base application) of Eisner et al. (1989). This fluoroprobe offers certain advantages over the other commonly used probe for pH_i 2′,7′-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF): (i) because of its two strong pH-sensitive peak emissions, SNARF displays a good signal-to-noise ratio for ratiometric recording at low light intensities; (ii) unlike BCECF, the dual emisson of SNARF requires no sequential mechanical switching of excitation filters, thus simplifying the epifluorescence set-up; (iii) because carboxy-SNARF-1 emission signals are at the yellow/red end of the visible spectrum, fluorescent drugs like amiloride, ethyl-isopropyl-amibride (EIPA), 4,4′-diisothiocyanostilbene 2,2′-disulphonic acid (DIDS) and cinnamate analogues do not interfere with the pH_i recording, even when used at high concentrations.