使用细胞外通量分析对透化细胞的呼吸活性进行综合测量。
Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis.
机构信息
Diabetes and Obesity Center, Institute of Molecular Cardiology, University of Louisville School of Medicine, Louisville, Kentucky, USA.
1] Diabetes and Obesity Center, Institute of Molecular Cardiology, University of Louisville School of Medicine, Louisville, Kentucky, USA. [2] Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky, USA.
出版信息
Nat Protoc. 2014 Feb;9(2):421-38. doi: 10.1038/nprot.2014.018. Epub 2014 Jan 23.
Extracellular flux (XF) analysis has become a mainstream method for measuring mitochondrial function in cells and tissues. Although this technique is commonly used to measure bioenergetics in intact cells, we outline here a detailed XF protocol for measuring respiration in permeabilized cells. Cells are permeabilized using saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), and they are provided with specific substrates to measure complex I- or complex II-mediated respiratory activity, complex III+IV respiratory activity or complex IV activity. Medium- and long-chain acylcarnitines or glutamine may also be provided for measuring fatty acid (FA) oxidation or glutamine oxidation, respectively. This protocol uses a minimal number of cells compared with other protocols and does not require isolation of mitochondria. The results are highly reproducible, and mitochondria remain well coupled. Collectively, this protocol provides comprehensive and detailed information regarding mitochondrial activity and efficiency, and, after preparative steps, it takes 6-8 h to complete.
细胞外流量 (XF) 分析已成为测量细胞和组织中线粒体功能的主流方法。虽然该技术常用于测量完整细胞中的生物能量学，但我们在这里概述了一种详细的 XF 方案，用于测量通透细胞的呼吸作用。使用皂素 (SAP)、溶血卵磷脂 (DIG) 或重组产气荚膜梭菌α毒素 (rPFO)（XF-质膜通透剂 (PMP) 试剂）对细胞进行通透化处理，并提供特定的底物来测量复合物 I 或复合物 II 介导的呼吸活性、复合物 III+IV 呼吸活性或复合物 IV 活性。还可以提供中链和长链酰基辅酶 A 或谷氨酰胺，分别用于测量脂肪酸 (FA) 氧化或谷氨酰胺氧化。与其他方案相比，该方案使用的细胞数量较少，并且不需要分离线粒体。结果高度可重复，并且线粒体仍然保持良好的偶联。总的来说，该方案提供了有关线粒体活性和效率的全面详细信息，并且在准备步骤后，完成整个实验需要 6-8 小时。
Zotero 插件