Increased and prolonged superoxide production in PTEN^−/− neutrophils. (A-E) Bone marrow–derived neutrophils (4 × 10⁵) from WT and PTEN^−/− mice were treated (A) with buffer containing no chemoattractant, (B) 500 nM fMLP, (C) 1 μM fMLP, (D) 10 μM fMLP, (E) 500 nM PMA, or (F) with (or without) 1 μg/mL LPS, 1 hour, 37°C, then 1 μM fMLP. ROS production was monitored in the presence of 50 μM isoluminol and 0.8 U HRP in a luminometer at 37°C. Chemiluminiscence (arbitrary light units) was recorded (for 2 seconds) at indicated time points. WT and PTEN^−/− neutrophils were assayed in parallel. Data are mean ± SD from 1 experiment representative of 3. (G) Bone marrow neutrophils (4 × 10⁵) from WT and PTEN^−/− mice were incubated with 1.5 mg/mL cytochrome-c (with or without 100 U/mL SOD) and stimulated with no chemoattractant, 100 nM fMLP, 1 μM fMLP or 10 μM fMLP. After 5 minutes, cytochrome-c reduction was detected by spinning down cells and reading absorbance (at 550 nm) of the supernatant in a spectrophotometer. Data are represented as the difference in absorbance between samples with and without SOD; mean ± SD from (n = 3) independent stimulations.