Validation of the MNGC assay (2 h post-infection). (A) Representative confocal images of RAW264.7 macrophages infected at 30 MOI with wild-type Bp K96243 (wt), or Bp ∆hcp1, or Bp ∆bsaZ respectively. Scale bar: 90 μm. Macrophages were infected with Bp for 2 h and then fixed, processed in IF and images were acquired and analyzed according to the MNGC analysis script (described in the Methods - Image acquisition and analysis section and shown in Figure 1B). (B) Bar graphs for the quantification of several cellular features of MNGC formation. (C) Bar graphs for the quantification of bacterial spots per MNGC cluster and total number of bacterial spots. In B and C means +/- SD are shown of 6 replicates per plate, 3 plates run on independent days (n = 18). For each replicate well >1000 nuclei were analyzed. **** p <0.0001; ** p < 0.01.