Supplementary Figure 4: Neutrophils extravasate adjacent to perivascular macrophages during E. coli infection.

(a) Figure depicts the methodology employed to calculate predicted neutrophil to macrophage correlation (detailed in Materials and Methods section). The total observed extravasation tracks for the blood vessel imaged are overlayed in comparison to the predicted tracks. The insets depict one such event of neutrophil extravasation (red) from blood vessel (blue, vessel wall marked in cyan) in relation to PVM (green, asterisk in main figure). 00:00, min:sec. (b) Time-lapse intravital multi-photon imaging of ear dermis of DPE-GFP mice infected with E. coli depicting the adherence and extravasation of adoptively transferred cells isolated from mT/mG mice (red, numbered 1-4) in close proximity to PVM (DPE-GFP, green). Lines (white) represent migration tracks of selected neutrophils (red) 00:00:00, hr:min:sec. (c) Statistical analysis of neutrophil extravasation site with respect to perivascular macrophages. Numbers of neutrophils extravasating at theoretical/random (white bars) or observed (black bars) distances from GFP⁺ PVM within the same vessels. Data is cumulative of 50 events of extravasation from 3 independent experiments. (d) Neutrophil influx, (e) ear thickness and (f) RBC counts of ears concomitantly injected with E. coli and Hla (10μg) or only E. coli alone for 12h p.i. Data shown are mean±SEM of 3 independent experiments. P-values were calculated using two-way ANOVA or two-tailed unpaired Student's t-test, **P<0.01; ***P<0.001; ****P<0.0001; NS, not significant.