A Toolbox to Profile Immunometabolism Tested in Macrophages

Macrophages are highly plastic immune cells that can adopt several activation states. Often, macrophages are stimulated in vitro with lipopolysaccharide (LPS) to elicit pro-inflammatory macrophages or interleukin (IL)-4 to obtain a homeostatic phenotype. Fundamental to these functional activation states is the regulation of cellular metabolic processes. The metabolic alterations underlying LPS- and IL-4-induced phenotypes have been described thoroughly with several -omics techniques. However, methods allowing for time- and cost-effective screening of cellular metabolism are rather limited. Here, we establish a comprehensive toolbox to assess cellular metabolism in a semi-high-throughput 96-well-plate-based format. We validated the approach on LPS- and IL-4-activated mouse and human macrophages by measuring extracellular flux, SCENITH, mitochondrial mass and membrane potential, glucose and lipid uptake, and mitochondrial substrate utilization. By discussing the experienced strengths, limitations and complementarities of the distinct techniques, we guide readers for efficient application of the toolbox in their immune subsets of interest.