Dicarboxy-dichlorofluorescein: A new fluorescent probe for measuring acidic intracellular pH

Abstract

Derivatives of fluorescein sensitive to pH are extensively utilized for the determination of intracellular pH (pH i). Available dyes have p K a values of approximately 7.0, and are not well suited for measuring acidic pH i. We examined the fluorescein derivative, 5 (and 6)-carboxy-2′,7′-dichlorofluorescein (CDCF) for its potential in the microspectrofluorometric measurement of pH i during acidic conditions. CDCF showed intense fluorescence and pH sensitivity near its “effective” p K a value of 4.2, using a 495 440 nm dual excitation wavelength ratio method. Protein interactions caused fluorescence ratio deviations which were most pronounced at the extremes of pH, whereas calcium and magnesium concentrations had little effect on the fluorescent ratio intensity. Intracellular calibration performed using nigericin in the presence of high potassium eliminated the need to correct for protein interactions, and the ratio method minimized any variations due to dye concentration differences or instrument fluctuation. Intracellular retention of the dye was high, and 95% of the initial signal remained after 1 h. Fluorescence bleaching was 14.5% after 1 h of continuous excitation and cell survival was not affected by dye loading. We conclude that CDCF is an excellent intracellular pH indicator in the pH range of 4–5.