Lipopolysaccharide lps

Blood was withdrawn under authorised supervision from six healthy donors who had given their informed consent. PBMCs were isolated from lithium heparin anti-coagulated blood by Ficoll-Paque™ Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) density gradient centrifugation and resuspended in VLE RPMI 1640 (Very Low Endotoxin) media (Biochrom AG, Berlin, Germany) supplemented with 10 % (v/v) FCS (Fetal Calf Serum), 2 mM L-glutamine (Sigma Aldrich, Chemie GmbH, München, Germany) and 1 % (v/v) penicillin/streptomycin (Biochrom AG, Berlin, Germany). 500 μl of a suspension of PBMCs (2 × 10⁶ cells/ml) were incubated in sterile 24-well plates (Multiwell™, Falcon®, Becton Dickinson Labware, NJ, USA) for 4 h at 37 °C and 5 % CO₂ with corn starch and the four different Holi colours (see above), respectively, each at 1.5 × 10⁶ particles/ml. The utilised concentration of the Holi colours and corn starch arose from dose-effect experiments that we performed with ambient dust (data not shown). Cells treated with PBS only (7.5 % v/v) served as negative control. Cells treated with LPS (Lipopolysaccharide) (Enzo Life Sciences, NY, USA) at 100 ng/ml served as positive control. Samples were centrifuged for five minutes at 300 g and supernatants were transferred to fresh cell culture plates and frozen at −20 °C until further analysed.

Bone marrow-derived macrophages were obtained from male and female C57BL/6 wildtype mice (5–7 months old), which were received from the animal facility of the University Hospital Frankfurt. The mice were euthanized and both legs together with the hip were extracted. Tibias, femurs and the hip were individually flushed with sterile PBS, the cell suspension was collected, centrifuged and the pellet resuspended in ACK buffer (Lonza) to remove erythrocytes. The obtained white pellet was resuspended in DMEM containing 20 ng/ml macrophage colony-stimulating factor (M-CSF) (Biotrend) and 4×10⁶ cells per well were seeded on a 6 well plate. After 7 days of differentiation macrophages were activated overnight in DMEM containing 25 ng/ml interferon-γ (IFN-γ) (PeProtech) and 100 ng/ml lipopolysaccharide (LPS) (Enzo Life Science) for M1 cells; and 25 ng/ml interleukin-4 (PeProTech) for M2 cells. DMEM was supplemented with 10% fetal calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).