Fig. 5

Neonatal HI and HT modulate composition of ex vivo sorted CD11b⁺cells. 1, 3 and 7 days after HI or sham operation, CD11b⁺cells were isolated via MACS from brains of uninjured sham-operated and HI-injured animals exposed to immediate HT or NT. Flow cytometry with a small aliquot of isolated cells was used to determine purity isolated cells (a, b) and to characterize the composition of sorted myeloid cells (c, d). Purity of CD11b⁺ cells was quantified as the percentage of living, i.e., propdidumiodide (PI) negative cells (a, b). According to Ly6G and CD45 expression, sorted cells were distinguished in resident microglia (red gate: CD11b⁺ CD45 ^int Ly6G⁻), peripheral monocytes/macrophages (orange gate: CD11b⁺ CD45^high Ly6G⁻) and neutrophils (green gate: CD11b⁺ CD45^high Ly6G ⁺) in living (i.e., PI negative) cells (c, d). Gates were set according to samples obtained prior to MACS. ***p < 0.001, n = 6 animals/timepoint for sham (left and right hemisphere pooled), n = 12 animals/timepoint for HI + NT and for HI + HT (2 ipsilateral hemispheres pooled per sample). Histograms and contour plots in a and (b) are representative for cells isolated 1 day after HI