42 Chapter 2 Different techniques are used to measure the metabolic status of cells, including extracellular flux analysis, colorimetric/fluorometric enzyme activity assays, and mass spectrometry (MS) based metabolomics and flux analysis (12). These techniques provide complementary information about the metabolic state of cells. We aim to clarify some of their advantages and disadvantages and provide an overview of when to use which method as well as how to combine them. Functional assays Widely used assays in the field of immunology are extracellular flux analyzers that give a functional readout of glycolytic or mitochondrial metabolic activity by measuring changes in energy metabolism in culture medium of cells (107). Mitochondrial function and respiratory capacity are assessed by measuring the oxygen consumption rate, which correlates with ATP-linked respiration, maximal and basal respiration, and proton leakage. It is a functional, real-time assay that does not measure the level of individual metabolites, but indirectly measures OXPHOS activity. Glycolytic activity can be assessed by using a glycolytic stress assay that forces the cells to use glycolysis by initial glucose starvation followed by glucose administration and ATP synthase inhibition. The extracellular pH is monitored to calculate the acidification rate. When glucose is converted to pyruvate and subsequently lactate during anaerobic glycolysis, H+ ions will be produced, shifting the pH of the medium. These assays have the advantage of being performed in living cells, but they are indirect indicators of changes in metabolic pathways or mitochondrial function, which play a pivotal role in macrophage activation and function as outlined above (108). However, individual metabolite levels or activity of individual enzymes are not quantified emphasizing the need for complementary analytical approaches to understand how metabolic pathways change during macrophage polarization. Enzymatic assays Key metabolites to follow with respect to the different macrophage phenotypes are listed in Table 1 and are part of glycolysis and the TCA cycle. The consumption rate of glucose, as the major source of energy for classically activated macrophages, gives an indication of the overall energy requirement. The conversion of glucose to pyruvate by the action of glycolytic enzymes is an indicator of the role of glycolysis and therefore it is of interest to measure some of its intermediates like glucose6-phosphate, fructose-6-phosphate or phosphoenolpyruvate. Since classically activated macrophages shift pyruvate conversion from acetyl-CoA production to
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