Evaluation of the frequency of internalization and intracellular persistence by IFC
The internalization frequency was evaluated in a cell culture model of infection in MG-63 osteoblasts at a multiplicity of infection (MOI) of 100:1, as previously reported (Bongiorno, Musso et al ., 2020). In this work we evaluated, with IFC, bacterial internalization after 3h p.i., and persistence after 24h p.i, as previously reported (Bongiorno, Musso et al ., 2020). We acquired 10,000 events for each sample and as quality control we used the stained and not stained bacterial suspension to exclude autofluorescence; a negative control of infected, but not permeabilized cells, was also acquired to guarantee the exclusively intracellular localization of green spots. Acquisition analysis was driven by the powerful INSPIREĀ® and IDEASĀ® packages (Amnis, EMD Millipore, Seattle, WA, USA).