Giuliano K A, Gillies R J
Department of Biochemistry, Colorado State University, Ft. Collins 80523.
Anal Biochem. 1987 Dec;167(2):362-71. doi: 10.1016/0003-2697(87)90178-3.
In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pKa of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load.
就准确性和灵敏度而言，细胞内捕获的、pH 依赖性荧光探针适合准确测量细胞内 pH。这些探针通常以酯化形式引入活细胞，其中游离酸通过酶促水解产生。酯和游离酸的荧光特性可能有显著差异，可能会出现光谱不确定性。我们在此描述了使用已刮涂加载到 BALB/c - 3T3 小鼠细胞中的 8 - 羟基芘 - 1,3,6 - 三磺酸（吡喃黄素）测量细胞内 pH 的方法。吡喃黄素的激发光谱对 pH 敏感，在 415 nm 处有一等吸收点，在 405 和 465 nm 处有峰值，它们分别随 pH 降低和升高。465/405 比值可用于监测 pH，而 415 nm 处的荧光表示剩余的总染料依赖性信号。刮涂加载的染料在细胞中可持续长达 6 小时。我们使用尼日利亚菌素/高钾在原位对这种染料进行了校准，发现该染料在原位的 pKa 为 7.82，而在体外为 7.68。我们观察到，当细胞受到急性碱性或酸性负荷时，它们可以将细胞内 pH 缓慢平衡至接近对照水平。
