Establishment of SCF/IL-3- and IL-3-dependent cells from C/EBPα^-/- FL in vitro. (A) C/EBPα^+/+ and C/EBPα^-/- FL cells were plated in cIMDM at 2 × 10⁵ cells/mL in the presence or absence of IL-3 or IL-3 plus SCF. Total viable cell numbers were determined over time by trypan blue exclusion and hemocytometer counting. (B) SCF/IL-3-2 and IL-3B cells were plated in proliferation assays in the presence or absence of 100 ng/mL SCF, 30 ng/mL IL-3, 50 ng/mL GM-CSF, 100 ng/mL human IL-7 (Peprotech), 100 ng/mL human IL-6 (Peprotech), 5 U/mL human erythropoietin (EPO), 50 ng/mL G-CSF (Amgen), 100 ng/mL human M-CSF (Peprotech), 500 U/mL murine interferon α (IFNα; Biosource, Camarillo, CA), or 50 mg/mL IL-4 (Peprotech) according to the procedures outlined in “Materials and methods.” (C) Cytocentrifuge preparations of cell clones (SCF/IL-3-2 and IL-3B) derived from these cultures were stained with Giemsa, and bright field photomicrographs were obtained at magnification × 400. (D) The expression of lineage-specific cell surface antigens was measured with use of fluorochrome-labeled antibodies and flow cytometry according to the procedures outlined in “Materials and methods.” Percentage positive for each marker was obtained by subtracting the background fluorescence of an isotype matched control.