Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines:: Comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-rosamine (H2-CMX-Ros) and MitoTracker Red 580 (MTR580)
Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines:: Comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-rosamine (H2-CMX-Ros) and MitoTracker Red 580 (MTR580)
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DOI：
10.1016/j.jim.2005.07.024
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发表时间：
2005-11-30
影响因子：
2.2
通讯作者：
Jayaraman, S
中科院分区：
文献类型：
--
作者：
Jayaraman, S
确定了细胞凋亡过程中T细胞白血病细胞系Jurkat和胰岛素分泌β细胞系NIT-1的线粒体电位变化的适用性，包括TMRE、H-2-CMX-Ros和MTR 580。新鲜收获的Jurkat和NIT-1细胞诱导进行凋亡显示差的电位敏感性，固有的荧光染料，TMRE的保留。用甲醛或多聚甲醛处理完全消除了两种细胞类型中的TMRE摄取，而不管细胞凋亡诱导。有趣的是，新鲜收获的凋亡Jurkat细胞表现出较低的保留H-2-CMX-Ros，表明在凋亡过程中淋巴细胞的氧化状态显着降低。这与NIT-1细胞相反，NIT-1细胞在失巢凋亡诱导后未能显示H-2-CMX-Ros保留的显著降低。多聚甲醛处理减少了活Jurkat细胞中H-2-CMX-Ros的保留，但仍允许区分保留H-2-CMX-Ros较差的凋亡细胞。然而，活的Jurkat细胞在甲醛处理后失去了保留H-2-CMX-Ros的能力。相比之下，用多聚甲醛或甲醛处理对H-2-CMX-Ros在活的和凋亡的NIT-1细胞中的保留没有显著影响。在T和β细胞系中，MTR 580的摄取不依赖于线粒体膜电位。然而，MTR 580与H-2-CMX-Ros在用甲醛固定和细胞透化后的凋亡Jurkat细胞的共聚焦显微镜分析中相当。这些数据表明，虽然TMRE和H-2-CMX-Ros适用于确定淋巴细胞凋亡期间线粒体膜电位变化，但只有TMRE适用于β细胞中的此类分析。H-2-CMX-Ros和MTR 580均被证明适用于线粒体的共聚焦成像。(c)2005 Elsevier B. V.保留所有权利。
The mitochondria-specific dyes, TMRE, H-2-CMX-Ros and MTR580 were determined for their suitability to measure mitochondrial potential changes of the T cell leukemia cell line Jurkat and insulin-secreting beta cell line NIT-1 during apoptosis. Both freshly harvested Jurkat and NIT-1 cells induced to undergo apoptosis displayed poor retention of the potential-sensitive, intrinsically fluorescent dye, TMRE. Treatment with formaldehyde or paraformaldehyde completely abolished TMRE uptake in both cell types regardless of apoptosis induction. Interestingly, freshly harvested apoptotic Jurkat cells exhibited lower retention of H-2-CMX-Ros, indicating marked reduction in the oxidative status of lymphoid cells during apoptosis. This is in contrast to NIT-1 cells which failed to display significant reduction in H-2-CMX-Ros retention after anoikis induction. Paraformaidehyde treatment reduced the retention of H-2-CMX-Ros in live Jurkat cells but still allowed the discrimination of apoptotic cells which poorly retained H-2-CMX-Ros. However, live Jurkat cells lost their ability to retain H-2-CMX-Ros after formaldehyde treatment. In contrast, treatment with paraformaldehyde or formaldehyde did not have significant impact on the retention of H-2-CMX-Ros in both live and apoptotic NIT-1 cells. The uptake of MTR580 was independent of mitochondrial membrane potential in both T and beta cell lines. However, MTR580 was comparable to H-2-CMX-Ros for confocal microscopic analysis of apoptotic Jurkat cells following fixation with formaldehyde and cell permeabilization. These data demonstrate that while TMRE and H-2-CMX-Ros are suitable for determining mitochondrial membrane potential changes during apoptosis in lymphoid cells, only TMRE is suitable for such analysis in beta cells. Both H-2-CMX-Ros and MTR580 proved to be suitable for confocal imaging of mitochondria. (c) 2005 Elsevier B.V. All rights reserved.