Transwell filter chemotaxis assay of guinea pig bone marrow eosinophils. A suspension of 3 × 106 bone marrow leukocytes was placed in the upper chamber, with eotaxin present in the lower chamber (▪) or upper chamber (#). Leukocytes that accumulated in the lower chamber were quantified using flow cytometry (FACScan; Beckton Dickinson). (A,B) Representative FACS dot-plot light forward scatter/side scatter profile of (A) mixed bone marrow leukocyte population placed into the upper Transwell chamber and (B) leukocytes migrated into the lower chamber in response to eotaxin (3 nmol/L, lower chamber for 30 minutes. Eosinophils shown in red). (C,D) May-Grunwald and Giemsa-stained cytospin preparations of (C) mixed bone marrow leukocyte population placed into the upper Transwell chamber and (D) leukocytes migrated into the lower chamber in response to eotaxin (3 nmol/L, lower chamber for 30 minutes). (E) Total eosinophils migrated in response to eotaxin present in the lower chamber (0 to 10 nmol/L) or upper chamber (3 nmol/L; #). Data represent the number of eosinophils migrated in 1 hour, mean ± SEM, using different cell preparations n = 6 to 12. No significant increase in eosinophil migration was observed when eotaxin (3 nmol/L) was added to the upper chambers. A significant difference between test and control groups is indicated by *(P < .05) or **(P < .01). (F) Time course of eosinophil chemotaxis induced by eotaxin (3 nmol/L, lower chamber). Data represent the number of eosinophils migrated at each time point, mean ± SEM for a single cell preparation performed in triplicate. The results shown are representative of three identical experiments. (•), Eotaxin 3 nmol/L; (○), Vehicle.
