A flow cytometric analysis of Gfi-1^+/− and Gfi-1^+/+ EML cells. Gfi-1^+/− and Gfi-1^+/+ EML cells were stained and analyzed using a FACSvantage flow cytometer and CellQuest software. (A) Cells (1.5 × 10⁵) were then labeled with conjugated antibodies for 20 minutes at 4°C. All antibodies (B220, Sca-1, CD34, c-kit, Mac-1, Gr-1, and Ter119) used for flow cytometry including matched isotype controls were conjugated to phycoerythrin (PE) and purchased from eBiosciences. Error bars indicate SEM. (B) Wright-Giemsa staining of EML^+/− and EML^+/+ cells following terminal neutrophil maturation. Myeloid differentiation of EML cells was conducted in IMDM medium containing 20% horse serum, IL-3, SCF, and 10 μM ATRA for 3 days. The cells were then transferred to IMDM medium containing 20% horse serum and GM-CSF. Within a week, GM-CSF–dependent EPRO cells emerged. These cells were terminally differentiated by the addition of 10 μM ATRA for 3 days. Cells were cytospun on the days indicated and subjected to Wright-Giemsa staining.