FLOW CYTOMETRIC MEASUREMENT OF LIPID-PEROXIDATION IN VITAL CELLS USING PARINARIC ACID
FLOW CYTOMETRIC MEASUREMENT OF LIPID-PEROXIDATION IN VITAL CELLS USING PARINARIC ACID
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DOI：
10.1002/cyto.990130704
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发表时间：
1992-01-01
期刊：
影响因子：
--
通讯作者：
CHOW, S
中科院分区：
文献类型：
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作者：
HEDLEY, D;CHOW, S
描述了一种使用时间分辨流式细胞术测量脂质过氧化的方法。由于其化学性质，天然荧光脂肪酸顺式parinaric酸很容易在脂质过氧化反应中被消耗。在37℃，5 μ m, 60'的条件下，它能以时间和浓度依赖的方式加载到中国仓鼠卵巢细胞中，荧光一致，明亮，无细胞毒性。荧光显微镜检查细胞表面和细胞膜呈弥漫性染色。在配备325nm hed激光器的Epics Elite流式细胞仪中检测细胞时，将细胞保持在37℃，并随时间测量405nm的parinaric酸荧光。氧化剂叔丁基过氧化氢的加入导致细胞内氧化的爆发，通过同时向细胞加载二氯荧光素来显示，并且随着时间的推移，parinaric荧光丧失。随后是细胞死亡，表现为前向光散射的丧失和碘化丙啶的吸收。用抗氧化剂α -生育酚200 μ m预处理细胞，降低了parinaric酸荧光的损失率，延缓了细胞死亡的发生。脂质过氧化分解产物丙二醛的同时生化测定证实，无论是否进行α -生育酚预处理，都与parinaric酸荧光的丧失有密切的时间关系，并表明流式细胞术检测脂质过氧化具有相当的灵敏度。线粒体染色剂十二烷基吖啶橙与花青素染料DiOC(6)3结合，顺式parinaric酸染色，后者可通过共振能量转移激发。由于这两种探针显示出一定程度的细胞器特异性，因此它们可用于测量在特定亚细胞位点由于脂质过氧化而导致的parinaric酸损失。尽管我们对该方法感兴趣的是检查氧化还原循环抗癌药物的作用和宿主抗氧化防御的完整性作为耐药性的可能机制，但它似乎是一种通用的技术，用于研究细胞损伤的重要过程，而这一过程很难用标准的生化分析来研究。
A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis-parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37-degrees-C, with 5-mu-M for 60' giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37-degrees-C while being examined in an Epics Elite flow cytometer equipped with a 325nm HeCd laser, and parinaric acid fluorescence at 405nm was measured over time. Addition of the oxidant tert-butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells with dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide.Pretreatment of the cells with the antioxidant alpha-tocopherol, 200-mu-M, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without alpha-tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity.The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis-parinaric acid staining and could be excited by the latter using resonance energy transfer. Because these two probes show a degree of organelle specificity, they can be used to measure the loss of parinaric acid due to lipid peroxidation at defined subcellular sites. Although our own interest in the method is to examine the actions of redox cycling anticancer drugs and the integrity of host antioxidant defences as a possible mechanism of drug resistance, it appears to be a versatile technique for investigating an important process of cell injury that is difficult to study using standard biochemical assays.