Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY 40536, USA.
J Neurosci Methods. 2011 May 15;198(1):36-43. doi: 10.1016/j.jneumeth.2011.03.007. Epub 2011 Mar 23.
The analysis of mitochondrial bioenergetic function typically has required 50-100 μg of protein per sample and at least 15 min per run when utilizing a Clark-type oxygen electrode. In the present work we describe a method utilizing the Seahorse Biosciences XF24 Flux Analyzer for measuring mitochondrial oxygen consumption simultaneously from multiple samples and utilizing only 5 μg of protein per sample. Utilizing this method we have investigated whether regionally based differences exist in mitochondria isolated from the cortex, striatum, hippocampus, and cerebellum. Analysis of basal mitochondrial bioenergetics revealed that minimal differences exist between the cortex, striatum, and hippocampus. However, the cerebellum exhibited significantly slower basal rates of Complex I and Complex II dependent oxygen consumption (p<0.05). Mitochondrial inhibitors affected enzyme activity proportionally across all samples tested and only small differences existed in the effect of inhibitors on oxygen consumption. Investigation of the effect of rotenone administration on Complex I dependent oxygen consumption revealed that exposure to 10 pM rotenone led to a clear time dependent decrease in oxygen consumption beginning 12 min after administration (p<0.05). These studies show that the utilization of this microplate based method for analysis of mitochondrial bioenergetics is effective at quantifying oxygen consumption simultaneously from multiple samples. Additionally, these studies indicate that minimal regional differences exist in mitochondria isolated from the cortex, striatum, or hippocampus. Furthermore, utilization of the mitochondrial inhibitors suggests that previous work indicating regionally specific deficits following systemic mitochondrial toxin exposure may not be the result of differences in the individual mitochondria from the affected regions.
线粒体生物能量学功能的分析通常需要每个样本 50-100μg 的蛋白质，并且在使用 Clark 型氧电极时每个运行至少需要 15 分钟。在本工作中，我们描述了一种利用 Seahorse Biosciences XF24 通量分析仪同时测量多个样本中线粒体耗氧量的方法，每个样本仅需 5μg 的蛋白质。利用这种方法，我们研究了从皮层、纹状体、海马体和小脑分离的线粒体是否存在区域性差异。分析基础线粒体生物能量学表明，皮层、纹状体和海马体之间几乎没有差异。然而，小脑的 I 型和 II 型复合体依赖的氧消耗的基础速率明显较慢（p<0.05）。线粒体抑制剂在所有测试样本中按比例影响酶活性，并且抑制剂对氧消耗的影响仅存在微小差异。研究鱼藤酮对 I 型复合体依赖的氧消耗的影响表明，暴露于 10 pM 鱼藤酮会导致氧消耗在给药后 12 分钟开始出现明显的时间依赖性下降（p<0.05）。这些研究表明，这种基于微孔板的线粒体生物能量学分析方法可有效地同时从多个样本中定量氧消耗。此外，这些研究表明，从皮层、纹状体或海马体分离的线粒体中几乎没有区域差异。此外，线粒体抑制剂的利用表明，先前表明系统性线粒体毒素暴露后区域性特定缺陷的工作可能不是受影响区域的个别线粒体差异的结果。
