FRET-based Ca2+ measurement in B lymphocyte by flow cytometry and confocal microscopy
FRET-based Ca2+ measurement in B lymphocyte by flow cytometry and confocal microscopy
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DOI：
10.1016/j.bbrc.2007.12.142
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发表时间：
2008-03-07
影响因子：
3.1
通讯作者：
Tsubata, Takeshi
中科院分区：
文献类型：
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作者：
Adachi, Takahiro;Tsubata, Takeshi
在B细胞抗原受体（BCR）连接后，诱导Ca 2+动员，这对于下游信号传导分子如MAP激酶的活化是必需的。尽管合成的荧光螯合剂如Fluo-4和Indo-1广泛用于BCR连接后的Ca 2+测量，但它们随着加载后的时间而泄漏或不利地定位于一些细胞器中。为了解决这些问题，我们引入了一种基因编码的荧光指示剂cameleon，它是一种基于荧光共振能量转移（FRET）的指示剂，包含两种荧光蛋白（CFP和YFP）和两种钙离子响应元件（钙调素（CaM）的变体和钙调素结合肽）。在这里，我们证明了cameleon以及常规的合成Ca 21指示剂能够通过流式细胞术在BCR连接后清楚地测量Ca 2+。此外，共聚焦显微镜分析允许我们检测BCR连接后单个细胞中基于cameleon的Ca 2+动员。(c)2007年爱思唯尔公司All rights reserved.
Upon the B cell antigen receptor (BCR) ligation Ca2+ mobilization is induced, which is essential for activation of downstream signaling molecules such as MAP kinase. Although synthetic fluorescent chelators such as Fluo-4 and Indo-1 are widely used for Ca2+ measurement upon BCR ligation, they are leaked or unfavorably localized into some organelles with time post loading. To solve these problems, we introduce a genetically encoded fluorescent indicator cameleon which is a fluorescence resonance energy transfer (FRET)-based indicator comprising two fluorescent proteins (CFP and YFP) and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). Here, we demonstrate that cameleon as well as a conventional synthetic Ca 21 indicator enables Ca2+ measurement by flow cytometry clearly upon BCR ligation. In addition, confocal microscopy analysis allows us to detect cameleon-based Ca2+ mobilization in a single cell upon BCR ligation. (c) 2007 Elsevier Inc. All rights reserved.