Imaging flow cytometry analyses in E13.5 fetal livers reveals 2 main blocks in erythropoiesis: one in Kit+ cells and the other in orthochromatic erythroblasts. (A, left) Gating strategies (of wild-type cells) used for imaging flow cytometry to quantitate Kit+ cells and progressive stages of terminal differentiation (for a more complete gating strategy, see supplemental Figure 1). Cells were stained with CD71, Kit, and DRAQ5/DAPI nuclear stain. BF indicates bright field images. (Right) Representative images of Kit+ (CFU-Es and proerythroblasts), basophilic (baso), polychromatic (poly), orthochromatic (ortho), and enucleating erythroblasts obtained using imaging flow cytometry. (B) Quantitation of median fluorescence intensity (MFI) of Kit, CD71, and Ter119 staining in Eklf^−/− fetal livers (n = 4 for kit and CD71; n = 2 for Ter119) normalized to Eklf^+/+ and Eklf^−/− (n = 8 for kit and CD71; n = 3 for Ter119). Ter119 expression is drastically reduced in Eklf^−/− erythroblasts during terminal differentiation. (C) Representative Eklf^+/+ and Eklf^−/− orthochromatic erythroblasts identified by imaging flow cytometry show the absence of Ter119 in Eklf^−/− cells. CD71 is in green, Ter119 in yellow, and DRAQ5 in red. (D) Alterations in relative proportions of live cells, erythroid cells that are Kit+, and progressive stages of terminal differentiation (basophilic, polychromatic, orthochromatic erythroblasts, and percentage of orthochromatic erythroblasts that were enucleating) were quantified using imaging flow cytometry for Eklf^+/+ and Eklf^+/− (n = 8), and Eklf^−/− (n = 4) E13.5 fetal liver cells. (E) Relative cell size (using CD71 area) and nuclear size (using DRAQ5/DAPI DNA stain area) of Eklf^−/− cells normalized to Eklf^+/+ and Eklf^+/− at progressive stages of differentiation. No significant differences were found between biological replicates of Eklf^+/+ and Eklf^+/− fetal livers (n = 8) vs Eklf^−/− fetal livers (n = 4). *P < .05, **P < .001, ***P < .0001.