Quantitative analysis of B. pseudomallei K96243 induced murine macrophage MNGC formation. (A) Representative 20X magnification confocal images of RAW264.7 macrophages that were not infected (mock) or infected with wild-type B. pseudomallei K96243 at a MOI of 30 at 10 h post-infection. Images of cell nuclei (stained with the Hoechst 33342 dye), cell cytoplasm (stained with CellMaskDR- CellMask DeepRed) and Bp: bacteria labeled using an anti-B. pseudomallei mouse monoclonal and a secondary anti-mouse/Alexa488 antibody. Scale bar: 90 μm. (B) Visual representation of the MNGC Image Analysis procedure. Each object (Nuclei) is pseudocolored with a unique color in the nucleus segmentation panel. Bacterial spots are pseudocolored in green in the spot segmentation panel. Nuclei clustering: Nuclei are clustered based on distance as described in Experimental procedures to generate the Cluster population. In the MNGC selection panel, image objects classified as MNGC are pseudocolored in green, and non-MNGC objects are pseudocolored in red. (C) Histograms representing the quantification of cellular attributes of the cluster population as measured by the MNGC image analysis procedure described in Figure 1B. (D) Histograms showing the results of the quantification of cellular attributes related to bacterial spot formation. In C and D means +/- standard deviation (SD) are shown for three independent B. pseudomallei macrophage infections performed on separate days and with six replicates/plate. n = 18 and > 500 nuclei were analyzed per well. **** p < 0.0001.