Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells.
Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells.
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DOI：
10.1152/ajpcell.1993.265.4.c1184
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发表时间：
1993-10
期刊：
影响因子：
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通讯作者：
T. B. Wiegmann;L. W. Welling;D. Beatty;D. E. Howard;S. Vamos;S. J. Morris
中科院分区：
文献类型：
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作者：
T. B. Wiegmann;L. W. Welling;D. Beatty;D. E. Howard;S. Vamos;S. J. Morris
使用新设计的荧光视频显微镜同时分析了暴露于各种刺激的 Madin-Darby 犬肾细胞和肾小球上皮细胞中细胞内钙浓度 ([Ca2+]i) 和细胞内 pH 变化之间的相互关系。细胞用 indo 1 和 SNARF 1 染料进行双重标记，并在 350 和 540 nm 下同时激发。对于两种比率染料，以 30 帧/秒的速度在 405、475、575 和 640 nm 下同时捕获四种发射波长的图像。 [Ca2+]i 未改变 SNARF 对 6.5 至 8.0 之间 pH 值的敏感性。 SNARF 比率图用于校正局部细胞钙计算中 pH 依赖性变化。 NH4Cl 负载产生了预期的碱化，同时 [Ca2+]i 上升。当 NH4Cl 被去除并且细胞变成酸性时，记录到 [Ca2+]i 的第二次升高。 [Ca2+]i 的两种变化均来自细胞内储存，因为它们在细胞外钙不存在的情况下持续存在。研究结果表明需要对 indo 1 记录进行 pH 校正。
The interrelationships between changes in intracellular calcium concentration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney cells and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence video microscope. Cells were double labeled with indo 1 and SNARF 1 dyes and were excited simultaneously at 350 and 540 nm. Images at four emission wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH between 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were used to correct the pH-dependent changes in the calculation of local cell calcium. NH4Cl loading produced the expected alkalinization and a concurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells became acidic, a second rise in [Ca2+]i was recorded. Both changes in [Ca2+]i were from intracellular stores since they persisted in the absence of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.