Jayaraman Sundararajan
Diabetes Research Institute, University of Miami School of Medicine, Miami, FL, USA.
J Immunol Methods. 2005 Nov 30;306(1-2):68-79. doi: 10.1016/j.jim.2005.07.024. Epub 2005 Sep 29.
The mitochondria-specific dyes, TMRE, H2-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 H2-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 H2-CMX-Ros retention after anoikis induction. Paraformaldehyde treatment reduced the retention of H2-CMX-Ros in live Jurkat cells but still allowed the discrimination of apoptotic cells which poorly retained H2-CMX-Ros. However, live Jurkat cells lost their ability to retain H2-CMX-Ros after formaldehyde treatment. In contrast, treatment with paraformaldehyde or formaldehyde did not have significant impact on the retention of H2-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 H2-CMX-Ros for confocal microscopic analysis of apoptotic Jurkat cells following fixation with formaldehyde and cell permeabilization. These data demonstrate that while TMRE and H2-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 H2-CMX-Ros and MTR580 proved to be suitable for confocal imaging of mitochondria.
研究了线粒体特异性染料TMRE、H2-CMX-Ros和MTR580在测量T细胞白血病细胞系Jurkat和胰岛素分泌β细胞系NIT-1凋亡过程中线粒体电位变化方面的适用性。无论是新鲜收获的Jurkat细胞还是诱导凋亡的NIT-1细胞，对电位敏感的固有荧光染料TMRE的保留能力都很差。用甲醛或多聚甲醛处理后，无论是否诱导凋亡，两种细胞类型对TMRE的摄取均完全被消除。有趣的是，新鲜收获的凋亡Jurkat细胞对H2-CMX-Ros的保留能力较低，表明淋巴细胞在凋亡过程中氧化状态明显降低。这与NIT-1细胞形成对比，NIT-1细胞在失巢凋亡诱导后，H2-CMX-Ros的保留能力没有显著降低。多聚甲醛处理降低了活Jurkat细胞对H2-CMX-Ros的保留能力，但仍能区分对H2-CMX-Ros保留能力差的凋亡细胞。然而，甲醛处理后，活Jurkat细胞失去了保留H2-CMX-Ros的能力。相比之下，多聚甲醛或甲醛处理对活的和凋亡的NIT-1细胞中H2-CMX-Ros的保留没有显著影响。在T细胞系和β细胞系中，MTR580的摄取均与线粒体膜电位无关。然而，在用甲醛固定和细胞通透处理后，MTR580在共聚焦显微镜分析凋亡Jurkat细胞方面与H2-CMX-Ros相当。这些数据表明，虽然TMRE和H2-CMX-Ros适用于确定淋巴细胞凋亡过程中的线粒体膜电位变化，但只有TMRE适用于β细胞的此类分析。H2-CMX-Ros和MTR580都被证明适用于线粒体的共聚焦成像。
