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-I ) 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 ratiometric 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.Key terms: Intracellular pH, proliferation, fluorescence intensity Cellular metabolism and function depends critically on maintenance of the intracellular (cytoplasmic) pH (pH,) within narrow limits. Measurement of pH, can therefore be used to obtain information about important processes in the cell, such as cell division (4,9). In contrast to other methods to detect changes in pHi, such as weak acid distribution and microelectrodes, the use of pH-sensitive fluorochromes in combination with flow cytometry allows analysis of heterogeneous cell populations on a single-cell basis.Human epidermis is a continuously renewing epithelium. Germinative cells in the lower layers are dividing in order to replace the dead cells in the upper layers, which are scaling off. In normal human epidermis not all of the germinative cells are cycling. We hypothesize that cell production is regulated by a G h G 1 induction of germinative cells (2). Methods to detect cycling cells, using the monoclonal antibody Ki-67 (3,171, anti-cyclin (7), or anti-BrdUrd (10,16) all have their own limitations. In this study our aim was the development of a flow cytometric method to measure ' pH, in quiescent and cycling keratinocytes using cultured keratinocytes as a model. We have tested the recently developed pH-sensitive dye SNARF-l(19) as a possible candidate to measure growth...

A simple optical fiber device for quantitative fluorescence microscopy of single living cells

“…6. This result is in agreement with those obtained by Van Erp et al (1991) and Martinez-Zaguilan et al (1991).…”

Cytotoxic effect of RB 6145 in human tumour cell lines: dependence on hypoxia, extra- and intracellular pH and drug uptake

“…The cell concentration was 1 x 106 cells ml-'. We used the fluorochrome carboxy-seminaphthorhodafluor-1 (C-SNARF-1) as the pH probe for our flow cytometry measurements (Bassnett et al, 1990;van Erp et al, 1991;Seksek et al, 1991). Its absorption spectrum is pH dependent, such that, once calibrated, the ratio of the absorbances at 575 and 640 nm gives an indication of the pHi as individual cells are analysed in the flow cytometer.…”

Flow cytometric measurement of [Ca2+]i and pHi in conjugated natural killer cells and K562 target cells during the cytotoxic process

“…From preliminary studies it was clear that leakage of this probe was negligible (data not shown), as was mentioned by Van Erp et al (13). Calibration of the cytosolar pH has been performed using high K + buffers with different pHs and nigericin (2 kg/ml) as the proton ionophore.…”