Abstract
The CCF4-AM Förster resonance energy transfer (FRET) assay is a sensitive approach to measure bacterial cytosolic translocation in live cells. The FRET pair hydroxycoumarin (donor) and fluorescein (acceptor) are linked by a CCF4-AM β-lactam ring, the substrate of β-lactamase. The exogenously added, neutral charged-FRET reagent can diffuse across the membrane and stay in the cytosol only once it is charged in the cytosol. When bacteria translocate from subcellular organelles (e.g., phagosomes) to the cytosol, the bacteria-associated β-lactamase cleaves the β-lactam ring, resulting in loss of FRET signal. Here we describe the fluorometer-based approach optimized for direct measurement of cytosolic translocation as a result of the EsxAB complex of Mycobacterium marinum in RAW264.7 cells.