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Ca 2+ is a ubiquitous ion involved in all known cellular processes. While global Ca 2+ responses may affect cell fate, local variations in free Ca 2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca 2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca 2+ signals induced by pathogens raises major technical challenges that need to be addressed. Here, we report protocols for the detection of global and local Ca 2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca 2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca 2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca<sup>2+</sup> Responses During <em>Shigella</em> Infection of Epithelial Cells