Figure 3.

Effect of properdin and thrombospondin repeats (TSR) 4+5 on the phagocytosis of Mycobacterium bovis BCG by THP-1 cells. (A) M. bovis BCG was treated with properdin at concentrations of 0, 2, and 20 µg/ml or with (B) TSR4+5 at concentrations 0, 1, and 5 µg/ml. The mycobacteria were incubated with macrophage for 2 h. After THP-1 cell lysis, surviving internalized M. bovis BCG were measured by plating lysates on 7H10 media to obtain colony-forming units (CFUs). The input value is the starting number of M. bovis BCG added to the THP-1 cells, before phagocytosis. A one-way ANOVA test was performed on the data to determine significant differences in CFU count by properdin or TSR4+5. All comparisons were significant (p < 0.05), unless where shown (ns, not significant, p > 0.05). Samples were analyzed in triplicate. (C) Differential uptake of GFP-M. bovis BCG by THP-1 macrophages after treatment with 0, 1, and 10 µg/ml of TSR4+5, or 10 µg/ml of BSA, used as a negative control. Cells were incubated for 2 h. Cells were then washed, fixed, and stained with AlexaFluor546-conjugated wheat germ agglutinin to reveal the plasma membrane (red), and the nucleus was stained with DAPI (blue). Images are shown as single sections, taken using a Leica DM4000 microscope; bar scale 10 µm.