Increased chemoattractant-induced ruffling in PTEN^−/− neutrophils. Neutrophils (2 × 10⁵) purified from WT or PTEN^−/− mice were plated on Labtek chamber slides and uniformly stimulated with fMLP or IL-8. (A) WT (left) and PTEN^−/− (right) neutrophils before (top) and 2 minutes after stimulation (bottom) with 100 nM fMLP. Scale bar, 25 μm. (B-C) Sensitivity analysis for chemoattractant-induced neutrophil ruffling. Neutrophils were uniformly stimulated with the indicated concentration of fMLP (B) or IL-8 (C), and the percentage of cells that ruffled or extended pseudopods was calculated from images captured 4 to 8 minutes after stimulus was added. Data are mean ± SD collected from (n = 3) separate preparations of neutrophils. *P < .05 versus wild-type neutrophils. Two videos of this experiment are included (Movie S1 and S2). (D-E) fMLP receptor levels in WT and PTEN^−/− neutrophils. Bone marrow–derived wild-type and PTEN^−/− neutrophils (0.5 × 10⁶) in HBSS/1%BSA/0.02% sodium azide were incubated with 3 nM, 6 nM, or 12 nM FITC-fMLP for 1 hour on ice, in the presence or absence of 10 μM unlabeled fMLP. Cells were then washed once and immediately analyzed by flow cytometry. Neutrophils were gated by using their characteristic forward/side scatter. (D) FACS analysis histograms of wild-type (left) and PTEN^−/− neutrophils (right).