Fig. 2

A revised gating strategy can be used to identify microglia in WNV-infection. a-d Quality control gates, including time (a), single cells (b), non-debris (c) and live cell (d) gates were applied before analysing cells. Neutrophils were also excluded by their expression of Ly6G (e). f Gating strategy one identifies a ‘resting’ microglia population (CD45^lo CD11b⁺) (R1: blue) and an ‘activated’ microglia population (CD45^int CD11b⁺) (R2: red). h, j Gating strategy 2, does not use ‘microglia-specific markers’ and identifies microglia as CX3CR1⁺ CD45^lo-int CD11b⁺ Ly6C^−/lo. Gating strategy 3 is a revised gating strategy which identifies microglia as CX3CR1⁺ CD45^lo-int CD11b⁺ P2RY12⁺ Ly6C^−/lo (h, i, l). n, o Gating strategy 4 uses a limited number of markers and identifies microglia as CD45^lo-int P2RY12⁺ CD11b⁺ CX3CR1⁺. g, k, m, p ‘Microglia’ populations gated using strategies 1 (g), 2 (k), 3 (m) and 4 (p), overlaid onto a tSNE plot, clustered on live cells from WNV dpi 7 brains. q, r Number (q) and frequency (r) of ‘microglia’ gated using strategies 1-4. Data is presented as mean ± SEM, from two independent experiments with at least six mice per group. **P < 0.0021, Kruskal-Wallis test and Dunn’s multiple comparisons test