Purification and separation of human neutrophils from whole blood promotes neutrophil activation and apoptosis. (A) Heparinized human blood or purified neutrophils were evaluated for the expression of l-selectin and CD11b in resting conditions. Expression of cell surface markers was analyzed by flow cytometry and shows mean fluorescence intensity (MFI) gated on CD66b-positive cells. Histograms show the representative MFI from the 5 independent donors (left). Graph shows MFI, ± standard error of the mean (right). **P < .0066; ***P < .0004. (B) MFI of phagosomal ROS was analyzed from neutrophils in blood and purified neutrophils using Fc-OxyBURST Green assay reagent after 15 minutes and with the addition of TNF-α or fMLP; n = 4. *P < .0152 (left); *P < .0133 (right); **P < .0019. (C) Apoptosis of neutrophils was analyzed by TUNEL assay from neutrophils in whole blood and purified neutrophils at 7 hours. All CD66b+ neutrophils (3000 cell events) were analyzed for TUNEL MFI; n = 3. **P < .0020. All statistical data analyzed by Student paired t test versus control values. fMLP, formyl-methionyl-leucyl-phenylalanine; ROS, reactive oxygen species; TNF-α, tumor necrosis factor α; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling.