Fig. S3

Fig. S3 Experiment to enumerate and characterize macrophages newly arrived to a granuloma within 24 hours. (A) Outline of injections. Bacteria are injected into the hindbrain ventricle at 30 hours post fertilization (hpf). In order to form infected lesions large enough to compare with WT bacteria, ∼5x the dose of ΔRD1 was used (50-100 in WT vs 250-500 in ΔRD1). Nascent brain granulomas usually appear two to three days post infection (dpi). At this point embryos with nascent granulomas are injected via tail vein with Hoechst 33342 nuclear dye, which stains the nuclei of cells in the blood and many tissues, but not the brain (data not shown). (B-C) Outline of counting and analysis. (B) At 24 hours after the Hoechst injection, the granulomas are imaged to quantitate the number of cells recruited. See Figure 1C-E. (C) To study the fates of arrived macrophages, a second set of granulomas was analyzed. Embryos were injected as before, but a subset of granulomas with no significant difference in arrival number were analyzed. (WT: 39.3+/- 13.6, ΔRD1: 30.0 +/- 12.99, p=0.312). (Analysis of this subset or of all granulomas yielded the same results). 3D DIC timelapse was used in addition to widefield fluorescence to allow positive identification of infected state. See Figure 2A-C. Collapsed nuclei have a distinct fluorescent appearance, but were cross-checked with DIC footage as well. See Figure 3D-E.