Characterization of RasGRP1-expressing cells in the bone marrow. (A) FSC and SSC of bone marrow cells from a representative Gf11^+/+ and Gfi1^−/− mouse showing the different distribution pattern (left). Distribution of bone marrow cells expressing high (hi), intermediate, and low (lo) levels of surface Gr1 showing a reduction of Gr1^hi and an increase in Gr-1^lo in a representative Gfi1^−/− mouse compared with control Gfi1^+/+ mouse. The percentage of cells within each gate is displayed (representative results from 5 experiments). (B) Cytospin preparations of sorted bone marrow Gr1^hi and Gr1^lo cell populations from a control Gfi1^+/+ mouse showing that RasGRP1-stained cells have the nuclear morphology of immature myeloid cells and are confined to the Gr1^lo cell populations. Mouse IgG1 isotype antibody was used as a negative control; DAPI shows nuclear morphology. RasGRP1 is undetectable in bone marrow Gr1^lo cell populations from Gfi1^−/− mice (representative results from 5 experiments). (C) Giemsa and DAPI staining of Gr1^lo-sorted bone marrow populations after immunostaining for intracellular RasGRP1 showing cellular morphology. RasGRP1 is detected in Gr1^lo bone marrow cell populations from a Gfi1^+/+ mouse but not from a Gfi1^−/− mouse. Representative images are shown. (D) Enlarged image from Giemsa stained Gr1^lo-sorted bone marrow populations showing the morphology of cells expressing RasGRP1 from the control Gfi1^+/+ mouse (top). The morphology of Gr1^lo cell populations from the Gfi1^−/− mouse that fail to display RasGRP1 immunostaining is shown (bottom) in representative images.