Generation of 5-HETE-PLs by human neutrophils in response to agonists. (A) Neutrophils were activated by ionophore (10μM), PMA (1 μg/mL), or fMLP (1μM) for 15 minutes. (B) Priming of 5-HETE-PE formation by stimulation with fMLP (1μM, 15 minutes) with 30 minutes of preincubation with cytochalasin B (10 μg/mL), granulocyte macrophage colony-stimulating factor (GM-CSF; 1nM), IL-8 (10nM), PAM₃CSK4 (1 μg/mL), LPS (1 μg/mL), or flagellin (0.1 μg/mL). (C) Activation by bacteria. Neutrophils (3 × 10⁶ cells) were incubated with fMLP (1μM) or live S epidermidis bacteria (S epi alone; 30 × 10⁶ cfu) or live S epidermidis (Opsin S epi; 30 × 10⁶ cfu) for 15 minutes. n ≥ 3; mean ± SEM data presented from 1 experiment and representative of 3; *P < .05, **P < .01, and ***P < .001 versus Control; #P < .05 and ##P < .01 versus fMLP alone. (D-H) Time course of activation with fMLP (1μM) after 30 minutes of preincubation with LPS (1 μg/mL). Formation of (D) free 5-HETE, (E) 16:0a/5-HETE-PC, (F) 18:0p/5-HETE-PE, (G) 18:1p/5-HETE-PE, (H) 16:0p/5- HETE-PE, and (inset) free 5-HETE. n ≥ 3; *P < .05, **P < .01, and ***P < .001 versus Control (0 minutes).