Figure 5. Necrosis induction does not lead to the release of mycobacteria from the phagolysosome to the cytosol.

THP-1 cells were infected with BCG at a MOI of 1 for 2 days before necrosis induction using 100 µg/ml concanavalin A for 24 h or 48 h. After CCF-4 loading for 2 h, cells were incubated 5 minutes in the presence of 2 µg/ml propidium iodide (PI) and then subjected to PFA fixation. Cells were imaged using fluorescence widefield Nikon Ti microscope with 40X objective (A). Picture acquisition was achieved randomly and automatically for each condition on 49 fields and further 450/535 nm intensity ratio measurements (B,C) were obtained through analysis by a specialized algorithm using the Metamorph software. The plots were representative of 2 independent experiments.