Intracellular signaling and cellular activation have been demonstrated to reside on multi-protein complexes rather than in isolated proteins. Consequently, techniques to resolve these complexes have gained much attention over the last few years. Förster Resonance Energy Transfer (FRET) coupled with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful tool to discriminate direct interactions between two proteins within a multi-protein complex. Here, we present the use of FRET-FLIM as an experimental tool for the interpretation of the inflammasome composition. We also introduce some considerations required for the correct use of this technique and the control experiments that should be implemented.
Keywords: Caspase-1; Endogenous proteins; FLIM; FRET; Immunofluorescence; Inflammasome; Macrophages; Multi-protein complex; NLRP3.