Flow Cytometric Analysis of Erythroblast Enucleation

Enucleation is the final step in mammalian erythropoiesis. In this process, the nucleus is extruded by budding off from the erythroblast, forming the reticulocyte. Herein, we describe the flow cytometry-based assays for enucleation assessment. The separation of nucleated erythroblasts, reticulocytes, and extruded nuclei by flow cytometry is based on DNA staining, surface expression of erythrocyte specific markers, or forward scatter (FSC). The enucleation of murine erythroblasts is assessed by the surface expression of murine erythrocyte marker Ter119 and DNA staining. Three discrete populations that represent nucleated erythroblasts, reticulocytes, and extruded nuclei are defined as Hoechst^medTER119^high, Hoechst^lowTER119^high, and Hoechst^highTER119^med, respectively. Another nuclei acid staining dye, SYTO16, is used for the assessment of human enucleation in combination with FSC. For human cells, the three populations that represent nucleated erythroblasts, reticulocyte, and extruded nuclei are identified as FSC^high SYTO16⁺, FSC^high SYTO16^-, FSC^lowSYTO16⁺, respectively.