Figure 1.

The effect of opsonization on phagocytosis of M. smegmatis by neutrophils was observed via confocal microscopy (A). Neutrophil cell membranes were stained with Wheat Germ Agglutinin (WGA, red) and GFP-expressing M. smegmatis was utilized. Yellow arrowheads show neutrophils that phagocytized M. smegmatis and white arrows point to uninfected neutrophils. The number of bacteria was counted in 100 neutrophils after 60 min of co-culture with either opsonized or non-opsonized M. smegmatis (B). Data are expressed as the number of neutrophils per number of internalized bacteria from three experiments. Internalization was also measured by Imagestream analysis where 2000 neutrophil events were collected and quantified based on GFP expression after either 15, 30, and 60 min of challenge with M. smegmatis at a multiplicity of infection (MOI) of 6 (C) or after 60 min at a MOI of 6, 50 or 100 (E). Representative images from the time course experiment are shown (D). Data for both Imagestream experiments are expressed as the mean ± SEM of the average %GFP neutrophils from three experiments. Two-way ANOVAs followed by Bonferroni post-hoc tests were used to determine significance.