Latex beads

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.

RAW264.7 cells were treated with PBS or simvastatin (0, 5, or 10 μM) for 8 h and incubated with latex beads (IgG-FITC complex) at a ratio of 1:100, according to the manufacturer's instructions (Cayman, Ann Arbor, MI). After incubation for 1 h, the treated cells were washed with PBS, fixed in 3.7% paraformaldehyde, and then subjected to flow cytometry analysis.