Fig. 3

Adoptively transferring, staining and blocking infiltrating monocytes support the use of our revised gating strategy. a Schematic showing the injection regimen used to block entry of monocytes in WNV-infected brains. Mice were treated intraperitoneally (i.p.) with a cocktail of blocking antibodies (anti-VLA4/CCL2/Ly6C) at dpi 5 and 6. b, c tSNE plot representing WNV dpi 7 brains from mice without blockade treatment (b) and with blockade treatment (c). d Number of microglia, Ly6C^hi, Ly6C^lo and microglia-like macrophages in WNV dpi 7 brains from non-treated animals and animals treated with isotype control antibodies or anti-VLA4/Ly6C/CCL2 blocking mAbs. Data is presented as mean ± SEM, with six mice per group, from two independent experiments. *P < 0.0332, **P < 0.0021, ***P < 0.0002, Kruskal-Wallis test and Dunn’s multiple comparisons test. e Schematic of adoptive transfer workflow. Ly6C^hi CD45⁺ CD11b⁺ CD115⁺ BM monocytes were sorted from WNV dpi 5 animals, stained with CFSE and transferred intravenously into matched WNV dpi 5 recipients. Brains were collected at dpi 7, and cells were isolated for FACs analysis. f FACs plot of live, CFSE⁺ cells from a recipient animal at WNV dpi 7. g CFSE⁺ cells overlaid onto a tSNE plot clustered on live cells from recipient animals at WNV-dpi 7. tSNE plot annotated with myeloid and lymphoid populations (h), showing that CSFE⁺, do not fall into the putative microglia population. i FACs plot showing the expression of CD45 and CD11b on CFSE⁺ transferred cells, microglia-like macrophages, Ly6C^hi macrophages and microglia. j Diagram showing the injection regimen used to stain cells infiltrating WNV-infected brains. Mice were injected intravenously with PKH67 at dpi 5, 6 and 7, 3 h prior to harvest. k tSNE plots representing WNV dpi 5, 6 and 7 brains from mice injected with PKH67, each generated separately. Microglial cells are circled in red and do not exhibit PKH67 staining