Neutrophil Activation and Phagocytosis Assay

To assess markers of neutrophil activation by flow cytometry, neutrophils were cultured with PRP in a ratio of 1:250 neutrophils: platelets (+ platelets). An equal amount of PPP was added to neutrophil-only cultures (- platelets). We have previously demonstrated that platelets exert their effect on leukocytes in a dose-dependent manner, and that this effect was most apparent at a neutrophil: platelet ratio of 1:250 [16 (link)]. In observance to this previous finding, we have employed the same neutrophil: platelet ratio in this study. To assess neutrophil phagocytosis, neutrophils were cultured either with WPs (+ platelets) or culture media (-platelets) and incubated with FITC-labelled rabbit IgG-coated latex beads (Cayman Chemicals, Ann Arbor, MI, USA) in a ratio of 1 μL of latex beads to every 200 μL culture media. For both neutrophil activation and phagocytosis, neutrophils ± platelets were left unstimulated or stimulated with 1 and 100 ng/mL of the following for 4 hours at 37°C/5% CO₂: LPS from Escherichia coli serotype R515 (a TLR4 agonist; Enzo Life Sciences, Farmingdale, NY, USA); Pam3CSK4 (a TLR2/1 agonist; Tocris Bioscience, Bristol, UK) and FSL-1 (a TLR2/6 agonist; Santa Cruz Biotechnology, Santa Cruz, CA, USA). LPS and FSL-1 were guaranteed by the respective manufacturers to be free of any contaminants that have agonist TLR activity.