线粒体功能障碍是许多类型神经退行性疾病的常见一级或二级因素，线粒体的质量、线粒体呼吸链复合物和线粒体DNA（mtDNA）拷贝数的变化通常在这些过程中出现。从人诱导多能干细胞（iPSCs）衍生的人类脑器官样结构（organoids）重现了大脑的三维细胞构架，并能够研究疾病机制和筛选复杂人体系统中的新治疗方法。本研究报道了一种基于流式细胞术的独特方法，用于测量iPSCs衍生的皮质脑器官样结构中的多个线粒体参数。该报告详细描述了从iPSCs生成皮质脑器官样结构的方案，生成的器官样结构的单细胞解离、固定、染色和后续流式细胞分析以评估多个线粒体参数。使用针对线粒体呼吸链复合物亚单位NADH：泛醌氧还酶亚单位B10（NDUFB10）或线粒体转录因子A（TFAM）的抗体与电压依赖的选择性阴离子通道1（VDAC 1）进行双重染色，可以评估每个线粒体中这些蛋白的含量。由于TFAM的数量与mtDNA的数量相对应，因此可以间接估计每个线粒体中mtDNA拷贝数。整个过程可以在2-3小时内完成。关键是，它能够同时量化多个线粒体参数，包括相对于线粒体质量的总水平和特定水平。

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain's three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.