In response to inflammatory signals, immune cells increase production of Nitric Oxide (NO), a soluble gas important for killing pathogens. Moreover, NO has other biological functions involved in signaling and metabolic regulation. Lately, the role of NO in immune cell reprogramming has been re-discovered and many metabolic changes that cells like macrophages undergo during proinflammatory conditions have been mechanistically ascribed to NO, which has become not only a regulator of Oxidative Phosphorylation (OXPHOS), but also a modulator of intracellular metabolism and the mitochondrial TCA cycle. Although some mechanisms have been established, understanding the diverse effects of NO and NO-derived reactive nitrogen species (RNS) on metabolic pathways and global immune cell metabolism is still challenging, considering the high sample amount required for these types of extremely important analyses in such growing field like immunometabolism. Integration of metabolomics and experiments with labelled carbons, oxygen probes, and chemical donors of NO-derived species allow for demonstration of precise interactions with different metabolic targets and for the framing of concentration and spatial necessities for its metabolic effects.

In this method collection, we aim to include topics that cover approaches in immunometabolism with a focus on NO. Our intent is to provide detailed procedures currently in use in the field for primary immune cell isolation, for metabolic analyses, and to introduce novel approaches to detect and quantify RNS. Therefore, the stoichiometries of RNS required for metabolic immunomodulation will be characterized, that could be then be translated into in vivo applications. Our collection will be a resource for anyone interested in the study of metabolism and mitochondrial dynamics in immune cells.