Neutrophil cell-surface adhesion molecule
LYSED, SEPARATION, ADHESION
Neutrophil cell surface adhesion molecules were assessed with antibodies staining for CD11b-PE (clone CBRM1/5; Affymetrix) and CD62L-FITC (clone DREG-56; BD Biosciences). Antibodies staining for CD14-Alexa Fluor 647 (clone MφP9; BD Biosciences) and CD16-eFluor 450 (clone CB16; Affymetrix) were used to facilitate separation of monocyte and neutrophil populations respectively. Heparinized whole blood (100 µl) was incubated with either wash buffer (unstimulated control), PMA (1.35 µM), or fMLP (0.83 µM) for 10 min at 37 °C followed by staining with antibodies for 20 min at room temperature in the dark. Erythrocytes were lysed with the addition FACS Lysing Solution (BD Biosciences). Cells were spun at 250×