Lps from escherichia coli serotype r515

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.

BMDCs were generated as described by Lutz et al. (48 (link)). Briefly, 2 × 10⁶ bone marrow cells from C57BL/6, C3H/HeN, or C3H/HeJ mice were seeded in 10-mm bacteriological culture dishes in RPMI (HyClone, Logan, Utah, USA) medium supplemented with 20 ng/mL mouse recombinant GM-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured for 10 days, after which they were collected via gently pipetting in preparation for incubation with hemocyanins. For most experiments, BMDCs were seeded at 1 × 10⁶ cells/mL in 6-well plates and incubated with the hemocyanins (1 mg/mL each) for 24 h. Then, the supernatants were collected to measure the cytokine levels, whereas the cells were treated with PBS supplemented with 10 mM EDTA and 4 mg/mL lidocaine and washed twice with FACS buffer in preparation for the maturation marker analysis by FACS. LPS from Escherichia coli Serotype R515 (Enzo Life Sciences) and LPS from Salmonella enterica serotype typhimurium (Sigma-Aldrich) and zymosan A (Sigma-Aldrich), were used as positive controls; PBS or unstimulated cells were used as a negative controls. The TLR2 control Pam3CysK4 was from Invitrogen Life Technologies (Waltham, MA, USA). The TLR9 control CpG oligonucleotide was from Oligos (Wilsonville, OR, USA).