Mendes Daniela, Silva Ana Maria, Oliveira Maria Manuel, Andrade Paula B, Videira Romeu A
REQUIMTE/LAQV, Laboratory of Pharmacognosy, Department of Chemistry, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.
Department of Life Sciences, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal.
Methods Protoc. 2023 Mar 24;6(2):32. doi: 10.3390/mps6020032.
Mitochondrial dysfunction and cytosolic oxidative stress are pathological biomarkers interlinked in several chronic diseases and cellular toxicity promoted by high-energy radiation or xenobiotics. Thus, assessing the activities of the mitochondrial redox chain complexes and the cytosolic antioxidant enzymes in the same cell culture system is a valuable approach to addressing the challenge of chronic diseases or unveiling the molecular mechanisms underlying the toxicity of physical and chemical stress agents. The present article gathers the experimental procedures to obtain, from isolated cells, a mitochondria-free cytosolic fraction and a mitochondria-rich fraction. Furthermore, we describe the methodologies to evaluate the activity of the main antioxidant enzymes in the mitochondria-free cytosolic fraction (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), and the activity of the individual mitochondrial complexes I, II and IV, as well as the conjugated activity of complexes I-III and complexes II-III in the mitochondria-rich fraction. The protocol to test the citrate synthase activity was also considered and used to normalize complexes. The procedures were optimized within an experimental setup to allow that each condition to be tested only requires sampling of one T-25 flask of cells 2D cultured, as the typical results presented and discussed here.
线粒体功能障碍和胞质氧化应激是几种慢性疾病以及由高能辐射或外源性物质引发的细胞毒性中相互关联的病理生物标志物。因此，在同一细胞培养系统中评估线粒体氧化还原链复合物和胞质抗氧化酶的活性，是应对慢性疾病挑战或揭示物理和化学应激源毒性潜在分子机制的一种有价值的方法。本文汇总了从分离细胞中获取无线粒体胞质部分和富含线粒体部分的实验步骤。此外，我们描述了评估无线粒体胞质部分中主要抗氧化酶（超氧化物歧化酶、过氧化氢酶、谷胱甘肽还原酶和谷胱甘肽过氧化物酶）活性的方法，以及富含线粒体部分中单个线粒体复合物I、II和IV的活性，以及复合物I - III和复合物II - III的共轭活性。还考虑并使用了测试柠檬酸合酶活性的方案来标准化复合物。这些步骤在一个实验设置中进行了优化，以使每个要测试的条件仅需对二维培养的一个T - 25细胞培养瓶进行取样，如此处呈现和讨论的典型结果所示。
