Schöller-Mann Alica, Matt Katja, Hochecker Barbara, Bergemann Jörg
Department of Life Sciences, Albstadt-Sigmaringen University of Applied Sciences, Sigmaringen, Germany.
Bio Protoc. 2021 Apr 5;11(7):e3980. doi: 10.21769/BioProtoc.3980.
Cellular health and function, as we know today, depend on a large extent on mitochondrial function. The essential function of mitochondria is the energy production, more precisely ATP production, via oxidative phosphorylation. Mitochondrial energy production parameters therefore represent important biomarkers. Studies on human cells have mainly been performed on cell cultures. However, peripheral blood mononuclear cells (PBMCs) are particularly suitable for such examinations. That's why this protocol describes a method to measure key parameters of mitochondrial function in freshly isolated PBMCs with the latest technology, the XF Analyzer. For this approach PBMCs are first isolated out of human anticoagulated blood. Next, they are attached to the surface of special microplates pre-coated with Poly-D-Lysine. During the subsequent measurement of oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) the stress reagents oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone and antimycin A are injected. Several mitochondrial parameters can be calculated from the results obtained. The application of this protocol allows the analysis of various influences, such as pharmaceuticals or environmental factors, on human cells.
正如我们如今所知，细胞的健康与功能在很大程度上取决于线粒体的功能。线粒体的基本功能是通过氧化磷酸化产生能量，更确切地说是产生三磷酸腺苷（ATP）。因此，线粒体能量产生参数是重要的生物标志物。对人类细胞的研究主要是在细胞培养物上进行的。然而，外周血单个核细胞（PBMC）特别适合此类检测。这就是本方案描述一种使用最新技术XF分析仪测量新鲜分离的PBMC中线粒体功能关键参数的方法的原因。对于这种方法，首先从人体抗凝血液中分离出PBMC。接下来，将它们附着在预先涂有聚-D-赖氨酸的特殊微孔板表面。在随后测量耗氧率（OCR）以及细胞外酸化率（ECAR）的过程中，注入应激试剂寡霉素、羰基氰化物4-（三氟甲氧基）苯腙（FCCP）、鱼藤酮和抗霉素A。从获得的结果中可以计算出几个线粒体参数。应用本方案可以分析药物或环境因素等各种因素对人类细胞的影响。
