Endothelium-interacting GFP⁺ leukocytes in the brain microvasculature are monocytes.

<p>PbA-infected MacGreen mice with NS were injected i.v. with 5 µg of either (<b>A</b>) an isotype control, (<b>B</b>) anti-Ly6C (n = 2) or (<b>C</b>) anti-Ly6G antibody (n = 2) 1 hour prior to mice undergoing intravital imaging of the brain microvasculature. Representative snapshots from each group are shown. Scale bars represent 30 µm in (A), 100 µm in (B) and 58 µm in (C). Blue represents SHG signals from extracellular matrix fibres. White arrows indicate direction of blood flow. To aid orientation, vascular walls are outlined. Cells stained with Ly6C antibody show an orange punctate stain that co-localises with GFP (red arrowheads = adherent cells; yellow arrowheads = rolling cells). A total of 37, 29 and 45 blood vessels were assessed for the isotype control, Ly6C and Ly6G groups respectively. Number of blood vessels per mouse: Isotype (n = 37), Ly6C (n = 14, 15) and Ly6G (n = 9, 36). Representative snapshots of intravital imaging of the dermis of Lysozyme-GFP mice after injecting 5 µg of (<b>D</b>) an isotype control, (<b>E</b>) anti-Ly6C or (<b>F</b>) anti-Ly6G antibody, as reported <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004236#ppat.1004236-Ng1" target="_blank">[34]</a>. Number of blood vessels per mouse: Isotype (n = 12), Ly6C (n = 11) and Ly6G (n = 11). Cells stained with Ly6C show a punctate stain as in the brain. Cells stained with Ly6G have a red surface stain. Scale bars represent 60 µm in (D), 30 µm in (E) and 20 µm in (F). (<b>G</b>) Changes in blood monocyte populations during ECM. Blood monocytes were quantified by flow cytometry after collection from either uninfected (UI) or PbA-infected mice that had developed ES or NS (n = 4–5 mice/group). Symbols represent individual animals and bars represent means. *P<0.05, **p<0.01 (ANOVA with Dunnett's post-test vs. UI control). (<b>H</b>) Representative flow cytometric plots showing blood monocyte populations in UI, ES and NS mice. The percentage of Ly6C^lo and Ly6C^hi monocytes are indicated. Data are from a single experiment (n = 4–5 mice/group).