Palmeira C M, Moreno A J, Madeira V M, Wallace K B
Department of Zoology, University of Coimbra, Portugal.
J Pharmacol Toxicol Methods. 1996 Feb;35(1):35-43. doi: 10.1016/1056-8719(95)00131-x.
We report a simple fluorometric method for the continuous monitoring of mitochondrial membrane potential and cell viability in suspensions of hepatocytes exposed in vitro to cytotoxic agents. Suspensions of freshly isolated hepatocytes (10(6) cells/mL) preloaded with rhodamine 123 (Rh 123, 100 mumol/L) are transferred to a thermostatically controlled mixed cuvette to which the desired cytotoxic agent is added. Rh 123 is a cationic fluorophore that is actively accumulated by cells in direct proportion to the mitochondrial membrane potential. Cell viability was estimated by monitoring propidium iodide (PI) fluorescence. Exposure of cell suspensions to the mitochondrial uncoupling agent FCCP caused an immediate and titratable increase in Rh 123 fluorescence. Subsequent treatment with digitonin did not change Rh 123 fluorescence, suggeseting that Rh 123 equilibrates rapidly across the intact cell membrane. Likewise, treatment of hepatocyte suspensions with inhibitors of mitochondrial respiration (rotenone, cyanide, or menadione) caused an immediate increase in Rh 123 fluorescence. This was accompanied by a progressive increase in PI fluorescence, suggesting a causal relationship between mitochondrial depolarization and cell injury. In contrast, 1,4-benzoquinone caused a time-dependent and linear increase in PI fluorescence that paralleled changes in Rh 123 fluorescence. Comparing the time courses for changes in PI and Rh 123 fluorescence suggests that for benzoquinone, the depolarization of the mitochondria is a consequence rather than a cause of the cell injury. This modified procedure provides a simple and specific technique for continuously monitoring mitochondrial membrane potential and cell viability in suspensions of freshly isolated hepatocytes. The advantage is that there is no need to separate cells from the incubation medium, making it possible to record real-time changes in mitochondrial membrane potential and cell viability throughout the in vitro exposure period.
我们报告了一种简单的荧光测定方法，用于连续监测体外暴露于细胞毒性剂的肝细胞悬液中的线粒体膜电位和细胞活力。将预先加载罗丹明123（Rh 123，100 μmol/L）的新鲜分离的肝细胞悬液（10⁶ 个细胞/mL）转移至恒温控制的混合比色皿中，并向其中加入所需的细胞毒性剂。Rh 123是一种阳离子荧光团，细胞对其主动摄取的量与线粒体膜电位成正比。通过监测碘化丙啶（PI）荧光来评估细胞活力。将细胞悬液暴露于线粒体解偶联剂 FCCP 会导致 Rh 123 荧光立即出现且可滴定的增加。随后用洋地黄皂苷处理并未改变 Rh 123 荧光，这表明 Rh 123 可在完整细胞膜上迅速达到平衡。同样，用线粒体呼吸抑制剂（鱼藤酮、氰化物或甲萘醌）处理肝细胞悬液会导致 Rh 123 荧光立即增加。这伴随着 PI 荧光的逐渐增加，表明线粒体去极化与细胞损伤之间存在因果关系。相比之下，1,4 - 苯醌导致 PI 荧光随时间呈线性增加，且与 Rh 123 荧光变化平行。比较 PI 和 Rh 123 荧光变化的时间进程表明，对于苯醌而言，线粒体去极化是细胞损伤的结果而非原因。这种改进的方法提供了一种简单且特异的技术，用于连续监测新鲜分离的肝细胞悬液中的线粒体膜电位和细胞活力。其优点是无需将细胞与孵育培养基分离，从而能够在整个体外暴露期间记录线粒体膜电位和细胞活力的实时变化。
