Allan Claire Y, Katsaros Tina, Missailidis Daniel, Fisher Paul R, Annesley Sarah J
Department of Microbiology, Anatomy, Physiology and Pharmacology, La Trobe University, Melbourne, VIC, Australia.
Methods Mol Biol. 2025;2920:203-223. doi: 10.1007/978-1-0716-4498-0_12.
Fibroblasts and peripheral blood mononuclear cells (PBMCs) are commonly utilized cell types for the analysis of mitochondrial function. Fibroblasts, derived from connective tissue, provide a reliable model for studying mitochondrial metabolism due to their active role in energy production and their accessibility for experimental manipulations. PBMCs, on the other hand, are a heterogeneous population of immune cells that include lymphocytes and monocytes. They offer the advantage of reflecting mitochondrial function in circulating cells and providing insights into systemic aspects of mitochondrial biology. Both cell types can be cultured and treated with various substrates or stressors to assess parameters of mitochondrial function. Here we describe the use of fibroblasts and PBMCs isolated from patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) to investigate mitochondrial abnormalities in the pathogenesis of this disease. Our techniques employ the use of fluorescent cellular dyes to measure mitochondrial mass, membrane potential and reactive oxygen species levels, luminescent measures of cellular NAD/NADH levels, and FRET-based measurements of the cellular and energy regulators, TORC1 and AMPK. These techniques are similarly useful for studying different physiological and pathological conditions.
成纤维细胞和外周血单核细胞（PBMCs）是常用于分析线粒体功能的细胞类型。成纤维细胞源自结缔组织，因其在能量产生中发挥的积极作用以及便于进行实验操作，为研究线粒体代谢提供了可靠模型。另一方面，PBMCs是包括淋巴细胞和单核细胞在内的异质性免疫细胞群体。它们具有反映循环细胞中线粒体功能并深入了解线粒体生物学系统层面情况的优势。这两种细胞类型都可以进行培养，并使用各种底物或应激源进行处理，以评估线粒体功能参数。在此，我们描述了使用从肌痛性脑脊髓炎/慢性疲劳综合征（ME/CFS）患者中分离出的成纤维细胞和PBMCs来研究该疾病发病机制中的线粒体异常。我们的技术利用荧光细胞染料来测量线粒体质量、膜电位和活性氧水平，利用发光测量细胞NAD/NADH水平，以及基于荧光共振能量转移（FRET）测量细胞和能量调节因子TORC1和AMPK。这些技术同样有助于研究不同的生理和病理状况。
