Figure 3

Chemotaxis and intracellular calcium flux in WT and HVCN1-deficient eosinophils. (A) The in vivo migration of neutrophils (left; n = 3 experiments) and eosinophils (right; n = 4 experiments) was determined by morphologically counting the cells collected from the peritoneal cavity of WT and HVCN1-deficient mice at the indicated time points following intraperitoneal injection of thioglycollate medium. Data are expressed as the mean ± SD with mice from each experiment indicated by different symbols. (B) Transwell migration assay was performed to compare the in vitro migration of WT and HVCN1-deficient eosinophils subjected to mEotaxin-1 at the indicated concentrations. The results are expressed as migratory index by determining the ratio of total cell number under mEotaxin-1 attraction to total cell number without mEotaxin-1. Data are representative of 3 experiments and expressed as mean ± SD. (C) Cytosolic Ca²⁺ response in WT and HVCN1-deficient eosinophils was determined with intracellular fluorescence dye Fluo-4 AM in the presence of 1 mM extracellular Ca²⁺. Shown are the representative overlaid traces out of 5 experiments for mEotaxin-1 (1-300ng/ml). The arrow indicates the time of mEotaxin-1 addition. The trace (blue: WT eosinophils; red: HVCN1-deficient eosinophils) represents the average fluorescence intensity from cultured BM-derived eosinophils at the collection speed of approximately 150 cells per second by flow cytometer Canto II. *, P < 0.05; **, P < 0.01; n.s., not significant.