Role of IL-5, IL-3, and GM-CSF in eosinophil development and survival. Bone marrow cells were initially stimulated with SCF/FLT3L for 4 days and further cultured in the presence of IL-5, IL-3, or GM-CSF. Cells were analyzed at indicated time points by flow cytometry. (A) Dot plots show the frequency of eosinophils (Siglec-F⁺SSC^hi) after 21 days in culture. (B) Eosinophil numbers are shown as mean + SD (n = 3). (C) Frequency of eosinophils (Siglec-F⁺SSC^hi) is shown as mean + SEM. Mature eosinophils were washed on day 12 after onset of culture and further cultured in the absence or presence of the indicated cytokines. Cells were harvested at the indicated time points and subjected to annexin V/propidium iodide (PI) staining for detection of apoptotic cells by flow cytometry. (D) Dot plots show representative stainings after 96 hours of culture. (E) Frequency of viable eosinophils (annexin V⁻PI⁻) shown as mean + SD (n = 3). SD, standard deviation; SEM, standard error of the mean.