DETERMINATION OF INTRACELLULAR PH OF BALB/C-3T3 CELLS USING THE FLUORESCENCE OF PYRANINE
DETERMINATION OF INTRACELLULAR PH OF BALB/C-3T3 CELLS USING THE FLUORESCENCE OF PYRANINE
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DOI：
10.1016/0003-2697(87)90178-3
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发表时间：
1987-12-01
影响因子：
2.9
通讯作者：
GILLIES, RJ
中科院分区：
文献类型：
--
作者：
GIULIANO, KA;GILLIES, RJ
在准确性和灵敏度方面，细胞内捕获的pH依赖的荧光探针适合于准确测量细胞内的pH。这些探针通常以酯化形式引入活细胞，其中游离酸是通过酶解产生的。酯和游离酸的荧光特性可能明显不同，可能会出现光谱不确定。在这里，我们描述了用8-羟基吡喃-1，3，6-三磺酸(吡喃)测定细胞内pH的方法。吡喃的激发光谱是pH敏感的，等色点位于415 nm，峰位于405和465 nm，随pH的升高而减小和增大。465/405的比值可以用来监测pH值，而415 nm处的荧光则表示剩余的总染料依赖信号。我们用黑素/高K+对该染料进行了原位校正，发现该染料的pKa为7.82，而在体外为7.68。我们观察到，无论是在急性碱性或酸性负荷下，细胞都可以缓慢地将细胞内的pH平衡到接近控制的水平。
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-hydroxypyrne-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.