199 Dominic De Nardo, Christine M. De Nardo (eds.), Innate Immune Activation: Methods and Protocols, Methods in Molecular Biology, vol. 1714, https://doi.org/10.1007/978-1-4939-7519-8_13, © Springer Science+Business Media LLC 2018 Chapter 13 Detecting Release of Bacterial dsDNA into the Host Cytosol Using Fluorescence Microscopy Roland Felix Dreier, José Carlos Santos, and Petr Broz Abstract Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular pat- terns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types. Key words cGAS, dsDNA, F. novicida, Innate immunity, Fluorescence confocal microscopy 1 Introduction Microbial pathogens, including viruses, parasites, and bacteria, constantly challenge eukaryotic organisms, which have thus evolved mechanisms of defense, collectively called immunity. The first line of host defense against invading microbes is the innate immune system, which has diverse germline-encoded pattern rec- ognition receptors (PRRs) that detect conserved pathogen- associated molecular patterns (PAMPs), allowing microbe phagocytosis and induction of inflammation [1]. In the cell, PRRs can have different subcellular locations, thus allowing a more pre- cise and regulated detection of PAMPs. For example, PRRs located on the plasma membrane or on endosomal membranes, such as Toll-like receptors (TLRs), typically recognize extracellular PAMPs. On the other hand, cytosolic PRRs, including NOD-like receptors Roland Felix Dreier and José Carlos Santos contributed equally to this work.