Stim1−/−neutrophils have normal adhesion and migration. (A) WT littermate chimera neutrophils (2 × 105 cells per well) were loaded with calcein AM plated on 0.2% milk– or different integrin ligand–coated plates in the presence of indicated stimuli with or without calcium for 15 minutes. Following 5 gentle washes, the fluorescence of the remaining adherent cells was determined. ***P < .001. (B) WT and stim1−/− chimera neutrophils (2 × 105) were plated on 0.2% milk– or different integrin ligand–coated plates, and adhesion was determined as above. (C) WT and stim1−/− neutrophils (106) were studied for chemotactic responses to MIP-2 (left) or fMLF (right) using 24-well transwell plates. The concentration of the stimuli in the lower wells is indicated. Migration was carried out for 45 minutes, and cells entering the lower chamber were counted by hemocytometer. (A-C) Data shown are representative of 3 to 4 independent experiments. (D) Left: WT and stim1−/− chimeras were injected intraperitoneally with 3% thioglycollate, and the number of neutrophils in peritoneal lavage fluid at 4 hours after injection was determined by flow cytometry (double staining for CD45.2/Gr-1). Right: peripheral blood was obtained from WT and stim1−/− chimeras at 4 hours following thioglycollate injection, and neutrophil counts were determined by using a HemaVet analyzer. Data shown are pooled from 2 independent experiments with at least 4 mice per experiment. (E) The footpads of either WT or stim1−/− chimeric mice were injected with 0.1 mL of 50 ng/mL MIP-2 and dissected 4 hours after injection and were formalin fixed and paraffin embedded. Tissue sections were stained with H&E. Photos are at ×40 magnification and are representative of 3 mice per group. ns, not significant.
