Effects of PI3K inhibitors on oxidase activity in fMLP-stimulated, TNF-α-primed human neutrophils. (A-C) Human neutrophils were primed with 200 U/mL TNF-α and were preincubated with 0 to 10 μM TGX-221 (A), IC87114 (B), and YM-024 (C) in the presence of luminol and HRP, as described in “Materials and methods.” Cells (3 × 10⁵ per well) were stimulated with fMLP (100 nM) and light emission (relative light units [rlu]) recorded at 10-second intervals using a Berthold MicroLumat Plus luminometer. All incubations were performed in duplicate or triplicate, and data (mean ± SEM or range) from 1 representative experiment of 2 to 5 experiments are shown. (D) Human neutrophils primed with 200 U/mL TNF-α (▴ and ▪) or vehicle (▵) were pretreated with either TGX-221 at the indicated concentrations (▴ and ▵) or TGX-221 in the presence of 1 μM IC87114 (▪), and the oxidative burst in response to fMLP (100 nM) was quantitated by reduction of cytochrome c. Data represent mean ± range of 2 separate experiments, with measurements performed in duplicate. (E-G) Human neutrophils primed with 200 U/mL TNF-α were preincubated with 0 to 30 μM IC87114 (E), YM-024 (F), or AS-252424 (G), and the oxidative burst in response to fMLP (100 nM) was quantitated by the reduction of cytochrome c. Data are mean ± SEM of 3 separate experiments, with measurements performed in duplicate.