FIG 3.

Host cells damaged by L. monocytogenes undergo Ca²⁺-dependent but K⁺-independent membrane resealing. Wild-type (wt) and LLO-deficient (Δhly) L. monocytogenes bacteria were incubated at various MOIs with HepG2 cells for 30 min at 37°C in standard medium (M1), Ca²⁺-free medium (M2), or high-K⁺ medium (M3). PI (100 μM) was added to the cell culture medium during the last 5 min of incubation. After fixation and DAPI labeling, phase-contrast (PC) and fluorescence images were acquired with a 20× objective. (A) Representative images. Images are displayed with the same scaling to show the relative intensities. Scale bar is 40 μm. (B and C) The average fluorescence intensity of PI was automatically measured in the nuclear region by quantitative fluorescence microscopy using Metamorph software. Results are expressed relative to the results for HepG2 cells incubated in M1 without bacteria. Results are the means ± SEM of at least three independent experiments performed in duplicate (*, P < 0.05; **, P < 0.01).