Abstract
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.
Highlights
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
Mean coefficient of variation (CV) of total protein concentration in wells for PBMC analysis was 46.8 ± 18.0%, 35.4 ± 19.5% and 46.1 ± 29.3% for PBMCs independently obtained from three different donors, A, B and C, respectively (Fig. 1c)
The relatively high CV for PBMCs as compared to RAW 264.7 macrophages and monocytes indicated that determination of total protein levels introduced higher variation between wells for PBMC XF assays than for XF assays with adherent RAW264.7 macrophages and monocytes, likely due to a higher loss of PBMCs compared to RAW 264.7 macrophages and monocytes after removal of XF assay medium and washing of the cells
Summary
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. Metabolic pathways do provide energy substrates and metabolic building blocks for immune cell proliferation, and dictate differentiation and effector functions of immune cells[1,2]. This dynamic and intimate crosstalk between cellular metabolism and immune cells has resulted in the emergence of the research field that is called immunometabolism[1,2]. Cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) are simultaneously measured in real-time in culture well-plates using fluorescent sensors in a Seahorse extracellular flux (XF) a nalyzer[32], and provides a powerful tool to study immune cell bioenergetics
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