Suspension cultures of human umbilical cord blood mononuclear cells supplemented with c-kit ligand-containing additives give rise to a mixture of cells belonging to several line-ages. Among those that differentiate in quantity are mature basophils, immature mast cells, and neutrophilic myelocytes. We used an ultrastructural immunogold method to detect the Charcot-Leyden crystal (CLC) protein, an eosinophil- and basophil-specific protein, to study cells that were obtained at sequential times from 3 to 14 weeks in culture. Basophils (and eosinophils, which were present in smaller numbers) labeled for the CLC protein; mast cells did not. The labeled basophil subcellular sites included formed intragranular, cytoplasmic and nuclear CLCs, cytoplasmic particle-filled and homogeneously dense granules, cytoplasm, nucleus, plasma membrane, and cytoplasmic and Golgi area vesicles. Individual basophil ultrastructural phenotypes similar to those associated with stimulated release and recovery reactions showed the expected variations in the gold-labeled subcellular compartments. Macrophages also were labeled for CLC protein within endocytotic-lysosomal structures; neutrophilic myelocytes did not contain CLC protein. On the basis of findings reported here, the combined ultrastructural morphology and immunogold phenotyping of cells differentiating in c-kit ligand-supplemented cultures allows accurate lineage assignment of the developing cells.
- Charcot-Leyden crystal protein
- Mast cells
- Neutrophilic myelocytes
- c-kit ligand
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