M. tuberculosis PknD triggers invasion of the brain endothelium. A. Brain endothelia were inoculated with either PknD sensor- or BSA-coated fluorescent microspheres, washed, and disrupted by trypsinization. Endothelia were subjected to flow cytometry and gated to remove extracellular microspheres not associated with whole cells. B. Flow cytometry analysis demonstrated that significantly more endothelial cells were positive for fluorescence when incubated with PknD sensor-coated microspheres compared to BSA-coated microspheres (7.7% vs. 0.6%; P = 0.0003). Cell counts are presented as mean ± standard deviation. C. Histograms show that discrete fluorescent-positive populations are evident in the cells inoculated with PknD sensor-coated microspheres, indicating that cell populations took up multiple quantities of microspheres. D. Microspheres were again pre-incubated with either custom anti-PknD serum or naïve serum, followed by inoculation onto endothelial cells. Pre-incubation with anti-PknD (1:250) significantly reduced the population of cells which were positive for fluorescent microspheres, compared to naïve serum, as is indicated in the figure by a horizontal bar (P = 0.001). Pre-incubation with anti-PknD (1:1250) had no effect on internalization, when compared to untreated cells (P = 0.07).