Fig. 1.

Eosinophils express Siglec-8 late in differentiation. 2.5×10⁶ of cord blood (CB) CD34⁺ cells were differentiated to the eosinophil lineage in the presence of SCF, Flt3-L, IL-3, and IL-5 for 3 days, followed by IL-5 only thereafter. 5×10⁵ cells were harvested at days 3, 6, 9, 12, 15, 18, and 21. Total RNA and cell lysate were prepared at each time point. Eosinophil differentiation was confirmed using eosinophil-specific Fast Green/Neutral Red staining. a Real-time quantitative PCR analysis of Siglec-8 mRNA expression during CD34⁺ cell differentiation into eosinophils. b Western blot analysis of Siglec-8 protein expression during CD34⁺ eosinophil differentiation using 2C4 monoclonal anti-Siglec-8 antibody. GAPDH was used as a loading control (lower panel). *The duplicate Day 21 sample is from an additional CB cultured under identical conditions