Human and Animal Physiology, Department of Animal Sciences, Wageningen University and Research, De Elst 1 6708 WD, P.O. Box 338, 6700 AH, Wageningen, The Netherlands.
Cell Biology and Immunology, Wageningen University and Research, P.O. Box 338, 6700 AH, Wageningen, The Netherlands.
Sci Rep. 2021 Jan 18;11(1):1662. doi: 10.1038/s41598-021-81217-4.
Analyzing metabolism of peripheral blood mononuclear cells (PBMCs) provides key opportunities to study the pathophysiology of several diseases, such as type 2 diabetes, obesity and cancer. Extracellular flux (XF) assays provide dynamic metabolic analysis of living cells that can capture ex vivo cellular metabolic responses to biological stressors. To obtain reliable data from PBMCs from individuals, novel methods are needed that allow for standardization and take into account the non-adherent and highly dynamic nature of PBMCs. We developed a novel method for extracellular flux analysis of PBMCs, where we combined brightfield imaging with metabolic flux analysis and data integration in R. Multiple buffy coat donors were used to demonstrate assay linearity with low levels of variation. Our method allowed for accurate and precise estimation of XF assay parameters by reducing the standard score and standard score interquartile range of PBMC basal oxygen consumption rate and glycolytic rate. We applied our method to freshly isolated PBMCs from sixteen healthy subjects and demonstrated that our method reduced the coefficient of variation in group mean basal oxygen consumption rate and basal glycolytic rate, thereby decreasing the variation between PBMC donors. Our novel brightfield image procedure is a robust, sensitive and practical normalization method to reliably measure, compare and extrapolate XF assay data using PBMCs, thereby increasing the relevance for PBMCs as marker tissue in future clinical and biological studies, and enabling the use of primary blood cells instead of immortalized cell lines for immunometabolic experiments.
分析外周血单个核细胞（PBMCs）的代谢为研究多种疾病（如 2 型糖尿病、肥胖症和癌症）的病理生理学提供了重要机会。细胞外流量（XF）测定法可对活细胞进行动态代谢分析，从而可以捕捉到 PBMC 对生物应激的体外细胞代谢反应。为了从个体的 PBMC 中获得可靠的数据，需要开发新的方法，这些方法允许标准化并考虑到 PBMC 非贴壁和高度动态的性质。我们开发了一种用于 PBMC 的细胞外流量分析的新方法，该方法将明场成像与代谢流量分析以及 R 中的数据集成相结合。使用多个血涂片供体来证明该方法具有较低的变异性，线性良好。通过减少 PBMC 基础耗氧率和糖酵解率的标准得分和标准得分四分位距，我们的方法可以准确、精确地估计 XF 测定参数。我们将我们的方法应用于 16 位健康受试者的新鲜分离的 PBMC，并证明我们的方法降低了组间 PBMC 基础耗氧率和基础糖酵解率的变异系数，从而减少了 PBMC 供体之间的变异性。我们的新型明场图像程序是一种强大、敏感且实用的归一化方法，可用于可靠地测量、比较和推断使用 PBMC 的 XF 测定数据，从而增加了 PBMC 作为未来临床和生物学研究中标记组织的相关性，并使原发性血细胞而不是永生化细胞系可用于免疫代谢实验。
