Characterization of bone marrow–derived basophils and mast cells. (A) Postsort analysis and nuclear morphology of purified basophils (IL-4/eGFP⁺c-Kit⁻) and mast cells (IL-4/eGFP⁺c-Kit⁺) after 11 days of culture of bone marrow cells in the presence of IL-3. (B) Titration of IL-3 and SCF to determine optimal culture conditions to generate basophils in vitro. Cultures were analyzed 11 days after setup. The experiment has been repeated with similar results. (C) Total numbers of basophils and mast cells on day 11 of culture in the presence of indicated concentrations of IL-3 and different concentrations of SCF (♦ indicates 0 ng/mL; ■, 0.03 ng/mL; ▴, 0.3 ng/mL; and ×, 3 ng/mL). The experiment has been repeated with similar results. (D) Semiquantitative RT-PCR of purified in vitro–generated basophils and mast cells to determine the expression level of different mast cell proteases. (E) Histamine content in sorted basophils (□) and mast cells (▩). The concentration was determined in 100 μL total cell lysate and normalized to 1000 cells. Bars show the mean plus or minus SD of triplicate wells. (F) Expression of different surface markers on mast cells (c-Kit⁺ cells) and basophils (c-Kit⁻ cells). Dot plots are gated on autofluorescence⁻IL-4/eGFP⁺ cells. For panels A, B, and F, the numbers in the quadrants indicate the frequency of each population.