Gilmore K, Wilson M
Department of Biological Sciences, University of Wollongong, Wollongong NSW, Australia.
Cytometry. 1999 Aug 1;36(4):355-8. doi: 10.1002/(sici)1097-0320(19990801)36:4<355::aid-cyto11>3.0.co;2-9.
A recent report by Macho et al. (Cytometry 25: 333-340, 1996) described the use of chloromethyl-X-rosamine (CMX-Ros) as a fixable probe for detection of loss of mitochondrial membrane potential (psi(mit)), an early event in many models of apoptosis. However, this previous report lacked a description of any direct comparisons between pre- and post-fixation analyses of normal and apoptotic cells stained with CMX-Ros.
Using a variety of cell types, we investigated the effect of paraformaldehyde fixation on cellular retention of CMX-Ros and the implications of this for the subsequent analysis of changes in psi(mit) in cells undergoing apoptosis.
We found that following fixation, the resolution between normal cells with polarized mitochondria and apoptotic cells with depolarized mitochondria is reduced to the extent that accurate discrimination between the cell types is no longer possible.
Overall, our results are consistent with CMX-Ros being a valid probe for psi(mit) in intact cells but only when the cells are stained and analyzed immediately. Thus, our results suggest that the proposed applications for CMX-Ros in multiple parameter analysis of fixed cells are inappropriate and will lead to spurious results.
马乔等人最近的一份报告（《细胞计数》25: 333 - 340, 1996）描述了使用氯甲基-X-若丹明（CMX-Ros）作为一种可固定的探针来检测线粒体膜电位（ψ(mit)）的丧失，这是许多细胞凋亡模型中的早期事件。然而，该先前报告缺乏对用CMX-Ros染色的正常细胞和凋亡细胞固定前与固定后分析之间任何直接比较的描述。
使用多种细胞类型，我们研究了多聚甲醛固定对CMX-Ros细胞保留的影响以及这对随后分析凋亡细胞中ψ(mit)变化的意义。
我们发现固定后，具有极化线粒体的正常细胞与具有去极化线粒体的凋亡细胞之间的分辨率降低到无法准确区分细胞类型的程度。
总体而言，我们的结果与CMX-Ros作为完整细胞中ψ(mit)的有效探针一致，但仅当细胞立即染色并分析时。因此，我们的结果表明，CMX-Ros在固定细胞多参数分析中的拟议应用是不合适的，将导致虚假结果。
