characterization of phagocytes delaminating from cystic YS-EBs(a) differentiation protocol schematic showing the suspension culture (top row), and the selective adherent conditions (bottom row). (b) low magnification view of delaminated lawn after plating stained for nuclear DAPI (grey scale), nuclear PU.1 (magenta) and membrane CD11b/ITGAM (green pseudocolor). Merged PU.1 and CD11b channels are depicted in the right panel. Scale bar: 25μm. (c) high magnification view of ramified cell in resting culture viewed under phase contrast (grey scale) and stained for nuclear PU.1 (magenta) and cytoplasmic IBA1/AIF1. Merged PU.1 and IBA1 channels are depicted in the right panel. Scale bar: 5μm (d, e) FACS scatter plots of harvested pMGLs for CD11b and IBA1 (left) or CD45 (right). (f) left panel, example image depicting the migratory path (white dashed arrow) of a single pMGL on a fluorescent bead lawn (yellow), as well as intracellular accumulation of phagocytosed fluorescent beads (arrow head) Scale bar: 10μm. Right panel, example image depicting a cotton fiber opsonized by pMGLs (scale bar: 25μm). (g) extracted frames from supplementary movie 2 depicting fluorescent beads (red arrowheads) taken up by a single pMGL Scale bar: 3μm. h: Quantification of pMGL EdU incorporation, measured at 2 weeks and 2 months (mean ± s.e.m. of 2 biological replicates, t-test, P<0.05).
