IL-3 is a hematopoietic growth factor, which is produced mainly by activated T cells. IL-3 is also secreted by other cell types, including mast cells, eosinophils, and keratinocytes. The broad spectrum of biological activities of IL-3 includes the stimulation of the proliferation and differentiation of immature pluripotent hematopoietic stem cells and various lineage-committed progenitor cells, leading to the production of most of the major blood cell types. In addition, IL-3 also affects the functional activity of mature mast cells, basophils, eosinophils, and macrophages.
Naive CD4+ CD62L+ T cells were isolated using the CD4+CD62L+ T Cell Isolation Kit II from mouse spleen cells and stimulated with CD3ε pure – functional grade and CD28 pure – functional grade antibodies in the presence of recombinant Mouse IL-2 and Mouse IL-4 and Anti-IFN-γ pure – functional grade antibody for four days. Then cells were harvested, washed, and incubated with or without PMA/Ionomycin for six hours. After two hours, brefeldin A was added. The cells were harvested, fixed, permeabilized, and intracellularly stained with Anti-IL-3 conjugated to PE (B) or APC (C), CD4-FITC and CD154-APC or CD154-PE. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer. Gating was performed according to the CD4 expression and side scatter properties of the cells. Cell debris was excluded from the analysis in a Propidium iodide versus Anti-IL-3-PE dot plot (A).
Analysis gate 1
Analysis gate 2