Subcellular fractionation of resting, FMLP-activated, and phagocytosing human PMNs. Distribution profiles of neutral sphingomyelinase and ceramide. Isolated and DFP-treated PMNs were resuspended at 2 × 10⁶/mL in PBS with CaCl₂(1 mmol/L) and MgCl₂ (1 mmol/L), and warmed to room temperature for approximately 1 hour. Cells were left untreated (control), stimulated with FMLP (100 nmol/L) for 10 minutes (FMLP), or stimulated with FMLP followed by addition of EIgG and incubation for 30 minutes (phagocytosis). Cells were resupended in disruption buffer, disrupted by nitrogen cavitation, and the postnuclear supernatant centrifuged on a two-layer Percoll density gradient. An equal number (ranging from 1.8 to 3.3 × 10⁸ cells) of control cells, FMLP-activated cells, and phagocytosing, FMLP-activated cells were fractionated in each experiment. The gradients were fractionated into 25 fractions by aspiration from the bottom of the tubes, and fractions assayed for neutral sphingomyelinase (SMase) activity, and every second fraction assayed for ceramide after removal of Percoll by ultracentrifugation. Results are the average of three experiments, normalized to a cell number of 3 × 10⁸.