Figure 4

No difference in uptake of mycobacteria between Cd36 ^+/+ and Cd36 ^-/- macrophages. A to F. M. marinum was biotinylated and incubated with Cd36 ^+/+ (top row) and Cd36 ^-/- (bottom row) macrophages for 3 hours to allow phagocytosis. Extracellular M. marinum was labeled using streptavidin-conjugated tetramethylrhodamine (TMR) and appears red. Macrophages were fixed and permeabilized and a second streptavidin-conjugated fluorophore (AlexFluor488(c)) was used to label intracellular M. marinum (appears green). A and B. Intracellular (green) M. marinum is readily visible within Cd36 ^-/- macrophages, with uptake qualitatively equivalent to Cd36 ^+/+ macrophages. C and D. Control conditions using cytochalasin D (10 μM) to inhibit phagocytosis, demonstrating decreased intracellular (green) mycobacteria in both Cd36 ^+/+ and Cd36 ^-/- macrophages. E and F. Control conditions using unbiotinylated mycobacteria, demonstrating the specificity of fluorescent labeling for mycobacteria. G and H. Image analysis demonstrates similar quantities of intracellular (G, green) and extra cellular (H, red) M. marinum (p = 0.29) in Cd36 ^+/+ and Cd36 ^-/- macrophages. I and J. Flow cytometry showed no significant difference in the fluorescence intensity associated with intracellular (green) or extracellular (red) M. marinum between Cd36 ^+/+ (solid line) and Cd36 ^-/- (dashed line). Shown for comparison are negative control conditions (unbiotinylated M. marinum, no fill color). K and L. In vitro, Cd36 ^+/+ and Cd36 ^-/- macrophages showed similar uptake of three mycobacterial species, M. tuberculosis (MOI = 10:1), M. bovis BCG (MOI = 10:1) and M. marinum (MOI = 1:1) (p > 0.05 for all species). Similar uptake was observed at different multiplicities of infection with M. tuberculosis (p > 0.05 for each MOI).