Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy‐SNARF‐1 and flow cytometry

Abstract

In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH‐sensitive dye carboxyseminaphthorhodafluor‐1 (SNARF‐1) as a possible candidate for flow cytometric pH measurements and compared it with 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) and 2,3‐dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF‐1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratio‐metric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7–8. With SNARF‐1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.