Calcium imaging of individual erythrocytes: Problems and approaches
Calcium imaging of individual erythrocytes: Problems and approaches
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DOI：
10.1016/j.ceca.2005.09.004
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发表时间：
2006-01-01
期刊：
影响因子：
4
通讯作者：
Lipp, P
中科院分区：
文献类型：
--
作者：
Kaestner, L;Tabellion, W;Lipp, P
尽管在红细胞中钙被认为在体内平衡中是重要的，但是由于细胞内环境的自发荧光或吸收特性，该离子浓度的测量通常被认为是相当有问题的。在这里，我们描述的实验，以评估流行的钙指标，如Fura-2，Indo-1和Fluo-4的可用性。在我们的实验中，Fluo-4被证明是更好的指示剂，因为（i）它的激发和发射特性受血红蛋白的影响最小，（ii）它是激发光不会导致红细胞显着自发荧光的唯一染料。从这些结果中，我们得出结论，使用指标，如Fura-2与红细胞一起必须重新审视。因此，我们在红细胞中使用Fluo-4，以证明前列腺素E-2和溶血磷脂酸刺激后这些细胞中的钙增加是稳健但不均匀的。对于后一种刺激，我们记录了单个红细胞的发射光谱，以确认基本上不变的Fluo-4发射，我们的结果强调，在红细胞内钙的测量是可靠的可能与Fluo-4和其他指标，特别是那些需要紫外线激发，出现不太有利。(c)2005爱思唯尔有限公司保留所有权利。
Although in erythrocytes calcium is thought to be important in homeostasis, measurements of this ion concentration are generally seen as rather problematic because of the auto-fluorescence or absorption properties of the intracellular milieu. Here, we describe experiments to assess the usability of popular calcium indicators such as Fura-2, Indo-1 and Fluo-4. In our experiments, Fluo-4 turned out to be the preferable indicator because (i) its excitation and emission properties were least influenced by haemoglobin and (ii) it was the only dye for which excitation light did not lead to significant auto-fluorescence of the erythrocytes. From these results, we conclude that the use of indicators such as Fura-2 together with red blood cells has to be revisited critically. We thus utilized Fluo-4 in erythrocytes to demonstrate a robust but heterogeneous calcium increase in these cells upon stimulation by prostaglandin E-2 and lysophosphatidic acid. For the latter stimulus, we recorded emission spectra of individual erythrocytes to confirm largely unaltered Fluo-4 emission, Our results emphasize that in erythrocytes measurements of intracellular calcium are reliably possible with Fluo-4 and that other indicators, especially those requiring UV-excitation, appear less favourable. (c) 2005 Elsevier Ltd. All rights reserved.