Fig. 4

Phagosome acidification in MOs that engulfed mycobacteria grown without phosphate. Phagocytosis assays were performed with MOs labelled with LysoTracker, whose red fluorescence intensifies at low pH, and FITC-labelled bacilli cultured with or without Pi. By immunofluorescence microscopy at least 400 MO were counted. MO emitting red fluorescence were scored as Lysotracker positive. The immunofluorescence patterns are shown; bacilli located in non-acidified compartments (LysoTracker negative) are green fluorescent. In acidified vacuoles (LysoTracker positive) bacilli emitted yellow fluorescence due to overlapping of FITC-labelled bacilli and Lysotracker red fluorescence (insert), (a). It was found that 80.75% of MOs incubated with mycobacteria grown with usual amount of Pi were LysoTracker positive while incubation with Pi-deprived bacilli 76.06% of MO were Lysotracker labelled (b), a difference that was not statistically significant (n s; Mann-Whitney Test). Four independent experiments were performed. FACS showed that the mean fluorescence index (MFI) was less in MO that phagocytosed Pi-deprived mycobacteria (c). This difference was not statistically significant (n s; Mann-Whitney Test)