Figure 3. Characterization of the phagocytic capacity of P. alecto primary and BM-derived DC and macrophages.

(a,b) Following 90 minutes co-culture with fluorescent FlashRed dye-conjugated polystyrene beads at +4 °C or at 37 °C, (a) primary (ex vivo) lung MNC and (b) 6 days-cultured BM-derived cells were analysed by flow cytometry to evaluate their phagocytic capacity. (a) Primary lung cells were gated first based on their expression of CD11b into CD11b⁻ lymphoid cells and CD11b⁺ myeloid cells, these two cell subsets being next gated based on MHC-II expression level into MHC-II⁻, MHC-II^int and MHC-II^hi (left panel), and analysed for their content in FlashRed beads. (b) 6 days cultured BM-derived cells were analysed as in Fig. 1b and their content in FlashRed beads was analysed. (c) Macrophages (CSF-1-MΦ) obtained from BM primary cells cultured in the presence of CSF-1 for 8 days (D8) were analysed for their phagocytic capacity of fluorescent FlashRed dye-conjugated polystyrene beads and analysed using the Amnis Imagestream. Histograms displaying the FlashRed intensity of macrophages cultured with beads at 4 °C (left histogram) or at 37 °C (right histogram) are displayed. Within histograms, the proportion of positive cells (gated as “positive”) is shown. Images of representative cells falling in the “negative” (upper right images) or in the “positive” (lower right images) gates of macrophages cultured at 37 °C are shown.