Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia,
Cell Physiol Biochem. 2020 May 29;54(4):556-566. doi: 10.33594/000000239.
BACKGROUND/AIMS: Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate.
We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine.
It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required.
The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na if calibration was carried out in the presence of ionophores.
背景/目的：钠是基本细胞功能的关键参与者。荧光探针是监测单个活细胞内钠离子水平的不可或缺的工具。由于细胞内钠离子敏感染料的荧光与水溶液中的荧光显著不同，因此使用离子载体间接原位校准荧光信号以平衡细胞内外离子浓度。比较使用荧光探针和直接火焰发射分析获得的数据的尝试很少，结果也不准确。
我们使用 Na 敏感探针 Asante Natrium Green-2（ANG）通过流式细胞术和标准火焰发射光度法，结合在 Percoll 梯度中通过细胞密度测定细胞内水来确定 U937 细胞中的细胞内钠离子浓度。通过已知的离子载体或通过哇巴因阻断钠泵或通过 staurosporine 诱导细胞凋亡来修饰细胞内 Na 浓度。
结果表明，当通过哇巴因介导的钠泵阻断或 staurosporine 诱导的细胞凋亡来修饰细胞内钠浓度时，两种方法都是可比的。用离子载体处理的细胞的 ANG 荧光比具有相同 Na 浓度但未用离子载体处理的细胞低约两倍。尽管其机制尚不清楚，但在需要定量评估细胞内钠离子浓度时，应考虑到这种影响。
钠离子敏感染料 ANG-2 是一种灵敏且有用的探针，可用于测定单个细胞和亚细胞微粒中 Na 含量和浓度的变化。如果在存在离子载体的情况下进行校准，则在不存在离子载体的情况下在研究细胞中测定的 ANG 荧光不能用作细胞内真实 Na 浓度的量度。
