Additional file 3 of High-parameter cytometry unmasks microglial cell spatio-temporal response kinetics in severe neuroinflammatory disease

Additional file 3 Microglia are easily identifiable in the homoeostatic brain. 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 (CD45lo CD11b+) (R1: blue) and an ‘activated’ microglia population (CD45int CD11b+) (R2: red). h, j Gating strategy two, does not use ‘microglia-specific markers’ and identifies microglia as CX3CR1+ CD45lo-int CD11b+ Ly6C-/lo. Gating strategy three is a revised gating strategy which identifies microglia as CX3CR1+ CD45lo-int CD11b+ P2RY12+ Ly6C-/lo (h, I, l). n, o Gating strategy four uses a limited number of markers and identifies microglia as CD45lo-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 mock-infected 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.