Expression of Runx1 in hematopoietic lineages analyzed by flow cytometry. (A) GFP expression was determined in hematopoietic cell subsets from wild-type (WT), Runx1-IRES-GFP^k/+, and Runx1-IRES-GFPk/k mice. The mean fluorescence intensity (MFI) of GFP expression in Runx1-IRES-GFP^k/+ and Runx1-IRES-GFP^k/k cells is shown in each panel. GFP expression in CD19⁺ B cells is shown; other hematopoietic subsets from Runx1-IRES-GFP^k/k mice likewise had an MFI approximately twice that of cells from Runx1-IRES-GFP^k/+ mice in all lineages that expressed Runx1. (B) Bone marrow (BM) cell suspensions were prepared from Runx1-IRES-GFP^k/k and wild-type mice, stained with either Ter-119, Mac 1 (CD11b), or GR-1, and analyzed for GFP expression by flow cytometry. In the analysis of Ter-119⁺ expression, the curve for Runx1-IRES-GFPk/k cells is superimposable with that of wild-type cells and is therefore not readily apparent. (C) GFP expression in lymph node T cells (CD3⁺) and B cells (CD19⁺) from wild-type and Runx1-IRES-GFP^k/k mice was determined using flow cytometry. In panels B-C, the filled and open curves indicate GFP expression in wild-type and Runx1-IRES-GFP^k/k cells, respectively. (D) Western blot analysis of FACS-purified CD3⁺ or CD19⁺ lymph node cells and Ter-119⁺ BM cells from Runx1-IRES-GFPk/k mice. The analyzed populations had more than 98% purity. Lysates from an equal number of cells for each group (6 × 10⁶ cells) were analyzed, and the blot was probed with a rabbit polyclonal antibody that specifically recognizes Runx1. Determination of the relative level of Runx1 mRNA expression by real-time RT-PCR in these FACS-purified cell populations is indicated below the Western blot. The results were normalized relative to input cell number.