Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

Microglia, the resident macrophage cells of the central nervous system, dynamically alter their metabolic programs in response to physiological and pathological cues. Understanding these metabolic shifts is crucial for elucidating their roles in inflammation. Here, we present a detailed protocol for assessing the glycolytic profile of primary microglia isolated from neonatal mouse brain cortices using a glycolysis stress test on an extracellular flux analyzer. This assay enables real-time measurement of ECAR, an indicator of glycolytic activity associated with low pH, such as lactate. Our approach involves treating cultured microglia under different conditions to examine how metabolic pathways are altered in response to various stimuli, including pro-inflammatory stimuli. Uniquely, our lab prepares fresh stock solutions of different reagents, including glucose, oligomycin, and 2-deoxyglucose (2DG), to target the different aspects of the pathway, with careful adjustment of pH for each reagent to ensure experimental accuracy and reproducibility. This method provides a robust platform for investigating glycolysis in primary microglia and offers insight into their functional states under inflammatory or disease-relevant conditions.