FLUORESCEIN CONJUGATES AS INDICATORS OF SUBCELLULAR PH - A CRITICAL-EVALUATION
FLUORESCEIN CONJUGATES AS INDICATORS OF SUBCELLULAR PH - A CRITICAL-EVALUATION
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DOI：
10.1016/0014-4827(84)90698-0
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发表时间：
1984-01-01
影响因子：
3.7
通讯作者：
GEISOW, MJ
中科院分区：
文献类型：
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作者：
GEISOW, MJ
异硫氰酸黄绿素（FITC）和异硫氰酸四甲基罗丹明（TRITC）与蛋白质或葡聚糖的缀合物用于测定亚细胞pH。6.8）。这使得pH值可以使用简单的Henderson-Hasslebach方程从光谱中准确得出。通过胞饮作用将FITC和TRITC缀合物递送至小鼠巨噬细胞溶酶体。然后以几种不同的方式测定溶酶体pH。首先，通过将亚细胞荧光光谱与自由溶液中获得的校准光谱直接匹配。其次，莫能菌素被用来平衡内部和细胞外的pH值。亚细胞的pH值，然后可以确定的FITC共轭物的荧光的相对增加，而不会损失的探针从溶酶体。这允许用探针原位校准pH依赖性。最后，允许巨噬细胞同时胞饮FITC和TRITC葡聚糖缀合物。pH值可以从溶酶体内2种染料的发射率确定。这些不同方法中的每一种都给出了相似的溶酶体pH值（4.8 ± 0.01）。0.1）。
Fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) conjugates to protein or dextran were used to determine subcellular pH. The pH dependence of fluorescence of FITC conjugates could be described by a single proton dissociation (pKa'' .apprx. 6.8). This allowed pH to be derived accurately from spectra using the simple Henderson-Hasslebach equation. FITC and TRITC conjugates were delivered to mouse macrophage lysosomes by pinocytosis. Lysosomal pH was then determined in several different ways. First, by direct matching of the subcellular fluorescence spectrum with calibration spectra obtained in free solution. Secondly, monensin was used to equilibrate internal and extracellular pH. Subcellular pH could then be determined by the relative increase in fluorescence of the FITC conjugate without loss of probe from the lysosomes. This allowed the calibration of pH dependence with the probe in situ. Finally, macrophages were permitted to pinocytose FITC and TRITC dextran conjugates simultaneously. pH could be determined from the ratio of emissions form the 2 dyes within the lysosomes. Each of these different methods gave a similar value for lysosomal pH (4.8 .+-. 0.1).