Enucleation is greater in MMP-low relative to MMP-high murine orthochromatic erythroblasts. (A) Enucleation rates (percentage of DRAQ5⁻ cells of total viable TER119⁺ cells) of freshly sorted, using the same flow cytometer, MMP-low and -high orthochromatic erythroblasts (G3) subjected to ex vivo enucleation assay. Progression of enucleation at multiple time points was measured in identical gates and plotted as fold changes normalized to time 0. (B) Representative ImageStream micrographs of nonenucleating, total enucleating, and late stage enucleating orthochromatic erythroblasts gated according to Δ_nucC = 0, 1 < Δ_nucC < 3, or Δ_nucC ≥ 3. BrightField (BF; gray), TER119 (erythroblast, blue), DRAQ5 (nucleus, red), and overlapping (white). (C) Medium Δ_nucC values of enucleating (Δ_nucC > 1) MMP-low and -high orthochromatic erythroblasts (n = 3 independent experiments). Cell doublets or dividing cells with 2 nuclei were excluded. (D) Violin plots of Δ_nucC values of enucleating cells (of panel C). Red dotted lines, medium; blue dotted lines, quartiles. Mean ± standard deviation (n = 3). *P < .05, **P < .01 Student t test.