Focusing on a population of enucleating cells. (A) Flow cytometry was used to quantitate enucleation efficiency. Erythroid cells (Ter119⁺) at the final stage of the long-term in vitro enucleation cultures were gated based on their size (FSC) and DNA content (Syto-16). The populations FSC^highSyto-16^high (purple), FSC^lowSyto-16^high (blue), and FSC^lowSyto-16^low (red) were sorted and examined by cytospins (B) shown to correspond to nucleated erythroblasts, nuclei, and enucleated red blood cells, respectively. Bar represents 10 μm. (C) A novel analysis in Imagestream^X (Amnis) was used to identify enucleating erythroblasts (here produced in a fast in vitro enucleation assay) and visualize the distribution of cytoskeleton and signaling molecules during enucleation. Erythroblasts were first gated according to size (Ter119 Area) and intensity of staining for Ter119 (mean pixel/area) into basophilic (BasoE), polychromatophilic (PolyE), and orthochromatic (OrthoE) erythroblasts, as previously described.²⁶  (D) Orthochromatic erythroblasts were then evaluated based on the following characteristics: “aspect ratio” of their shape (ratio of the minor axis/major axis) in bright-field image and “Delta centroid Ter119-Draq5,” calculated as the distance of the center of the Ter119-labeled erythroblast or reticulocyte from the center of the Draq5-labeled nucleus. The population of small cells with high Ter119 expression that also exhibited a low aspect ratio and high δ centroid Ter119-Draq5 (gated here by red elliptical) was enriched in enucleating cells. (E) Representative images of cells, stained with phalloidin–AlexaFluor-488, Ter119-PE, and Draq5, are shown along with their corresponding dot-points on the flow cytograms in panels C and D. F-actin was visible to form a CAR, shown by white arrowheads in subpanels Eiv, Ev, and Evi, in the cleavage furrow between incipient reticulocyte and pyrenocyte.