LPS isolation and macrophage stimulation

LPS was extracted using LPS extraction kit (iNtRON Biotechnology, UK), according to the manufacturers’ instructions. To eliminate protein contamination, treatment with protease K was performed prior to the extraction steps. The protease K (AppliChem, Germany) (20 mg/ml) was added to the cell mixture and incubated at 56 °C for 1 h. The efficiency of the LPS preparation was determined by Limulus Amebocyte Lysate (LAL chromogenic Endotoxin Quantitation kit, ThermoFisher) to measure the LPS concentration, according to the manufacturers’ instructions.

THP-1 human cells (American Type Culture Collection, ATCC TIB-202™) were cultured with the medium (RPMI-1640 medium supplemented with 10 % foetal calf serum, HEPES, l-glutamine, ampicillin and streptomycin) (Sigma-Aldrich) in a humidified cell culture incubator with 5% CO₂ at 37 °C. THP-1 cells were differentiated into macrophage cells by stimulation with phorbol myristate acetate (PMA). Cells were centrifuged and resuspended in fresh supplemented RPMI-1604 medium to a concentration of 8 × 10⁵ cells per ml. Phorbol myristate acetate (10 ng ml⁻¹) was added to diluted cells, and then aliquoted into 24-well plate by adding 1 ml of cell suspension per well. The differentiation of THP-1 monocytes were complete after 48 h of incubation, as exemplified by induction of adherent cells phenotype under the microscope.

Phorbol myristate acetate and any non-adherent cells were removed by replacement of the medium with fresh medium prior to addition of serial concentrations of LPS (4.5, 0.45, 0.045, 0.0045, 0.00045 ng/ml) to the appropriate wells in triplicate. Differentiated THP-1 cell cultures without LPS served as the negative controls. The wells are thoroughly mixed and incubated at 37 °C for 24 h. The samples were collected at 4, 6, 8 and 24 h and stored at −20 °C. The production of macrophage-derived cytokines (IL-6 and TNF-alpha) were analysed using DuoSet^® ELISA kit (R&D systems), and cytokine concentrations were calculated according to the manufacturers’ instructions.