Polarization of PSGL-1 in neutrophils migrating toward a chemoattractant gradient or on endothelial cells.

(A) Neutrophils were allowed to adhere to FN80-coated coverslips for 10 minutes. C5a was then deposited at one edge of the bottom of the well (lower right-hand corner of photograph) and cells were allowed to migrate for 5 minutes. Cells were fixed and stained with anti-PSGL-1 KPL-1 mAb (green fluorescence). Epifluorescence and bright-field conditions were photographed on the same frame by means of double exposure. (B) Neutrophils were allowed to adhere to an endothelial cell monolayer activated by TNF-α for 5 minutes at 37°C. Cells were then fixed and double stained with anti-ICAM-3 TP1/25 mAb (green fluorescence) and anti-ICAM-1 MEM111 (red fluorescence) mAb. (C) Neutrophils were allowed to adhere to an endothelial cell monolayer activated by TNF-α for 5 minutes at 37°C. Cells were then fixed and stained with anti-PSGL-1 KPL-1 mAb. Epifluorescence and bright-field conditions were photographed on the same frame by means of double exposure.