In vivo generation of basophils from bone marrow progenitors by IL-3 treatment. (A) In vitro generation of bone marrow–derived basophils. Bone marrow cells were cultured with 10 ng/mL of IL-3 for 7 days. At the end of culture, cells were harvested and stained for FcϵRI and c-kit. FcϵRI/c-kit double positive cells represent mast cells, while FcϵRI single positive cells represent basophils. Relative proportions are indicated. (B) In vivo generation of basophils by IL-3. C57BL/6 wild-type mice were implanted with a miniosmotic pump containing 5 μg IL-3. Seven days after pump implantation, cells from bone marrow, liver, spleen, and lymph nodes were harvested and stained for CD49b and FcϵRI. Proportions of CD49b⁺ FcϵRI⁺ cells (basophils) are indicated. No basophils were found after PBS pump implantation (data not shown). (C) Phenotypic analysis of IL-3–generated basophils. Basophils obtained by IL-3 pump implantation were analyzed for the expression of Thy1.2 and 2B4 (filled histogram plots), surface molecules known to be expressed on basophils. Open histogram plots represent isotype control antibody staining. (D) Cytospin samples of basophils isolated from indicated tissues by cell sorting were stained for Wright-Giemsa staining. Images were acquired using an Olympus BX51 microscope equipped with a 40×/0.75 numerical aperture objective lens (Olympus, Center Valley, PA). Images were taken using an Olympus DP70 digital camera with DP controller software. The images were subsequently processed with Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA).