Visualizing Leishmania Phagosome Dynamics

Macrophages cultivated in the HiQ4 multichamber dishes and infected with DsRed2-expressing L. amazonensis for 24 hours were incubated with 200 nM of Lysotracker Red DND-99 probe (Invitrogen) for a pulse of 30 min, washed and given fresh medium in the microincubator coupled to the confocal unit. The dynamic measurement of PV volumetric enlargement was performed as described [28 (link)], acquiring 10 microscopic fields per experimental condition. PV volumes in μm³ in each analyzed macrophage were graphically represented by a color scale applied to each PV isosurface, ranging from cyan (smaller) to magenta (larger PV). PV volume isosurfaces were also obtained from Dil-ox-LDL fluorescence for correlations between PV size and ox-LDL PV accumulation, using the same methodology. Similar to volume, cell sphericity is a measure obtained from three-dimensional image reconstructions assessed as described [87 (link)].