Perez-Terzic C, Stehno-Bittel L, Clapham D E
Department of Pharmacology, Mayo Foundation, Rochester, MN 55905, USA.
Cell Calcium. 1997 Apr;21(4):275-82. doi: 10.1016/s0143-4160(97)90115-9.
The fluorescent indicator Fluo-3 is widely used to monitor the calcium concentration ([Ca2+]) in the cytoplasm and nucleus of various cells. Estimates of nuclear [Ca2+] are based on the assumption of identical behavior of Fluo-3 in different cellular compartments. The assumption is not valid if the fluorescence properties of the dye are altered by the nuclear environment, independent of the [Ca2+]. To determine the effects of the nucleoplasm on the behavior of Fluo-3, we applied laser scanning confocal microscopy and spectrophotometry to measure fluorescence intensity as well as emission and absorbance spectra of the Ca2+ indicator, Fluo-3. Spectra were measured in intact Xenopus oocytes, neuroblastoma cells, and cytoplasmic and nucleoplasmic homogenates. The fluorescence signal in intact cells loaded with Fluo-3 was approximately 2-times higher in the nucleus when compared to the cytoplasm. The fluorescence intensity of Fluo-3 in nucleoplasmic homogenates was higher than in cytoplasmic homogenates or internal buffers even when [Ca2+] was clamped. Despite identical [Ca2+], pH, and temperature, the emission and absorbance spectra of Fluo-3 from nuclear homogenates displayed a higher fluorescence at each wavelength measured when compared to spectra from cytoplasmic homogenates or internal buffer solutions, and saturated above 100 nM. These findings demonstrate that the composition of the nucleoplasm changes the fluorescence properties of the calcium indicator Fluo-3. Consequently, analysis of nuclear calcium dynamics must take into account the distinct behavior of Fluo-3 in different cellular compartments.
荧光指示剂Fluo-3被广泛用于监测各种细胞的细胞质和细胞核中的钙浓度（[Ca2+]）。对细胞核[Ca2+]的估计基于Fluo-3在不同细胞区室中行为相同的假设。如果染料的荧光特性因核环境而改变，而与[Ca2+]无关，那么该假设就不成立。为了确定核质对Fluo-3行为的影响，我们应用激光扫描共聚焦显微镜和分光光度法来测量Ca2+指示剂Fluo-3的荧光强度以及发射光谱和吸收光谱。光谱是在完整的非洲爪蟾卵母细胞、神经母细胞瘤细胞以及细胞质和核质匀浆中测量的。与细胞质相比，加载了Fluo-3的完整细胞中，细胞核中的荧光信号大约高2倍。即使[Ca2+]被钳制，核质匀浆中Fluo-3的荧光强度也高于细胞质匀浆或内部缓冲液中的荧光强度。尽管[Ca2+]、pH和温度相同，但与细胞质匀浆或内部缓冲液溶液的光谱相比，核匀浆中Fluo-3的发射光谱和吸收光谱在每个测量波长处都显示出更高的荧光，并且在100 nM以上达到饱和。这些发现表明，核质的组成改变了钙指示剂Fluo-3的荧光特性。因此，对细胞核钙动力学的分析必须考虑Fluo-3在不同细胞区室中的不同行为。
