149 5 General Discussion polymerase chain reaction (RT-qPCR) to investigate the expression of genes linked to a macrophage anti-inflammatory phenotype. Cellular Metabolism: Investigating macrophage metabolism is crucial for understanding their functional state. Methods like Seahorse XF analysis or 13C-glucose tracing can assess parameters like mitochondrial respiration, glycolysis, and fatty acid oxidation. These measurements help in deciphering the bioenergetic changes occurring in activated macrophages. In Chapter 4 the extracellular flux analysis was used to investigate how the metabolism of alveolar-like macrophages changed when they were seeded on different surfaces and in Chapter 3 to explore how it might change when treated with an inflammatory stimulus (LPS) combined with anti-inflammatory agents (KDACis). This technique allows to measure oxygen concentration (Oxygen Consumption Rate (OCR)) and pH (Extra Cellular Acidification Rate (ECAR)) in the media of live cells in real-time, therefore indirectly measuring energy metabolism. Enzymatic Activity: Studying enzymatic activity can reveal how these enzymes are regulated during macrophage activation and their contribution to metabolic processes like glycolysis, oxidative burst, and lipid metabolism. In Chapter 4 the activity of glycolytic enzymes was measured in alveolar-like macrophages that were either seeded on plastic or on collagen-coated plastic. Metabolite Analysis: Metabolomics allows researchers to analyze the metabolic profile of macrophages. Changes in metabolites like glucose consumption, lactate production, or lipid metabolism can indicate how macrophages are responding to inflammation. Metabolite analysis provides insights into their energy requirements and metabolic adaptations during activation. In Chapter 3 gas chromatography– mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) with fluorescence detection were used to measure metabolites in the context of alveolar-like macrophages stimulated with LPS and treated with KDAC inhibitors. Major TCA cycle metabolites and the TCA cycle substrate amino acids were quantified. Other techniques that we have not applied in our manuscripts but that are of relevance and described in Chapter 2 include measuring surface markers or antigens that can be detected by techniques such as flow cytometry or immunohistochemistry, the visualization of cellular morphology using microscopy techniques, or methods using functional assays such as phagocytosis assays, oxidative burst assays, and chemotaxis assays.
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