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. 2004 Jun;72(6):3204–3217. doi: 10.1128/IAI.72.6.3204-3217.2004

FIG. 3.

FIG. 3.

Quantitation of immunofluorescence staining for CD63 in THP-1 cells infected with F. tularensis RCI or LVS. Monolayers of human THP-1 cells were incubated with latex beads, formalin-killed F. tularensis, or live F. tularensis LVS or RCI as described in the text. Monolayers were washed, incubated for an additional 1 to 16 h, fixed, permeabilized, stained for CD63 and F. tularensis by using immunofluorescence reagents, and examined by confocal and two-photon laser scanning fluorescence microscopy, and the numbers of bacteria and latex beads that colocalized with CD63 were enumerated. Colocalization of the F. tularensis RCI and LVS with CD63 reached a maximum of 60 to 70% 2 to 4 h after infection. Thereafter, with continued intracellular multiplication, the colocalization of F. tularensis with CD63 steadily declined, falling to 15% by 16 h. Latex beads and formalin-killed F. tularensis colocalized extensively (70 to 95%) with CD63 at all time points. This experiment was performed twice, with similar results. Similar results were also obtained with LAMP1 and LAMP2 and when MDM were used.