van Erp P E, Jansen M J, de Jongh G J, Boezeman J B, Schalkwijk J
Department of Dermatology, University Hospital, Nijmegen, The Netherlands.
Cytometry. 1991;12(2):127-32. doi: 10.1002/cyto.990120205.
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 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.
在本研究中，我们描述了一种使用流式细胞术测量培养的人角质形成细胞内pH值的方法。角质形成细胞带来了一个技术问题，因为该群体在大小和代谢活性（非特异性酯酶活性）方面存在异质性，导致染料摄取存在差异。为了弥补这一点，选择了随pH值变化颜色的染料。记录两个波长处的荧光强度比值，并通过参考质子离子载体尼日利亚菌素存在下的pH值，将其用作细胞内pH值的度量。然而，迄今为止发表的方法通常不会将比率技术与488 nm氩离子激光激发相结合。因此，我们测试了最近开发的pH敏感染料羧基半萘罗丹明氟-1（SNARF-1）作为流式细胞术pH测量的可能候选物，并将其与2',7'-双（羧乙基）-5,6-羧基荧光素（BCECF）和2,3-二氰基对苯二酚（DCH）在发射光谱、分辨率、范围和细胞荧光稳定性方面进行了比较。SNARF-1具有488 nm而非紫外光的实用且稳定的激发波长，它基于实际发射位移提供了比率测量的可能性，并且在7-8的pH范围内具有卓越的分辨率。使用SNARF-1，我们发现与高血清培养（6%）相比，在低血清条件（0.2%）下培养的角质形成细胞中，细胞内pH值相对较低的细胞比例更高。此外，pH值变化伴随着相对DNA含量的变化。这些发现表明，细胞内pH值可能是人类角质形成细胞早期功能增殖的标志物。
