5-HETE-PLs are associated with membrane fractions and not released after activation of neutrophils. Cells were activated with LPS and fMLP. Neutrophils were centrifuged to separate cells from extracellular supernatant before lipid extraction and analysis. (A) 5-HETE-PLs were retained in the cell pellet. (B) Free 5-HETE is both retained in the cell and released into the extracellular media. n ≥ 3; mean ± SEM data presented from 1 experiment and representative of 3. (C) After lysis by N₂ cavitation, neutrophils were fractionated by centrifugation before lipid extraction and analysis with LC/MS/MS; mean ± SEM data are pooled from 3 independent experiments.