Chemotx plate

Human granulocytes were obtained by ficoll separation and erythrocyte lysis with 1× red blood cell lysis solution (Miltenyi Biotec, Bergisch Gladbach, Germany). After washing twice with PBS, cell viability and cell count were assessed by standard 0.5% (w:v) trypan blue cell staining (Biochrom, Berlin, Germany). For chemotaxis experiments, the wells of ChemoTx^® Plates (NeuroProbe, Gaithersburg, USA; 96-well plates, with 5 -µm pore size, 5.7 -mm filter in diameter, 300 -µL capacity) were filled with 300 µL of chemotactic reagents dissolved in migration buffer (Hanks' solution with 20 mM HEPES and 0.1% BSA at pH = 7.6 (all purchased from Sigma-Aldrich, Taufkirchen, Germany)). Chemotactic reagents PGE₂, PGE₃, LTB₄, and LTB₅ were all purchased from Cayman Chemicals (Ann Arbor, USA), which were initially dissolved in ethanol and then diluted with migration buffer to 1000 nM. Eosinophilic cells were diluted to 6 × 10⁶ cells/mL in migration buffer, and 50 µL of the cell suspension (300.000 cells) were placed on top of the filters. After 2 h at 37 °C incubation time, cells on the top of the filter were discarded, and cells in the solutions of the lower chambers were counted for 60 s with a rapid flow rate using the FACSVerse flow cytometer (BD Biosciences, Heidelberg, Germany) and the FACSuite software v1.0.6 (BD Biosciences, Heidelberg, Germany).

Human monocytes were treated with demeclocycline (10 μM). After 1 h of incubation at 37°C with 5% CO₂, IFNγ (100 ng/ml)/IL-1β (100 ng/ml) was added. After 24 h, human monocytes were harvested and resuspended in RPMI 1640 media supplemented with 2% penicillin/streptomycin, 10% fetal bovine serum, L-glutamine, and 1 mM sodium pyruvate. Two hundred thousand cells were plated onto the filters of 5 μm pore size ChemoTx plates (NeuroProbe). Recombinant human CCL2 (Peprotech) (10 ng/ml) was diluted in supplemented RPMI 1640 media and 300 μl/well was added into wells below the filter so as to provide a chemotactic stimulus. Two controls were used in this assay. The first control was medium only. The second one was chemokinetic control where the cells plated onto the filter contained the 10 ng/ml of CCL2 as in the underlying well. To obtain a standard curve, halving numbers of cells were plated ranging from 0 to 200,000. Cells were incubated at 37°C in humidified air with 5% CO₂ for 16 h. They were then washed off the top of the filter and the plate spun at 1,400 rpm for 10 min at room temperature. One hundred and fifty microliter of the media was discarded in the microplate and replaced with 15 μl of alamarBlue® (Invitrogen). The plate was then placed at 37°C in humidified air with 5% CO₂ for 4 h and signal was read at 570 nm. This assay was also conducted with mouse BMDM.

Migration of MOLT-4 cells towards chemokine was assayed using 96-well ChemoTx plates with 5 µm pores (Neuro Probe, Gaithersburg, MD, USA). Chemokine (mCCL25) at 100 nM was incubated in the presence of increasing amounts of recombinant proteins, as indicated in the bottom wells, for at least 15 min at 37 °C. Cells (2.5 × 10⁵ per well) previously washed and resuspended in 0.1% FCS in RPMI1640 medium were carefully laid on the top filter and allowed to migrate for 2–6 h at 37 °C. After this period, nonmigrated cells from the top filter were rinsed away with phosphate buffered saline (PBS), and the number of migrated cells in each well determined using the Cell Titre Aqueous One Solution viability assay (Promega, Madison, WI, USA). The number of cells was extrapolated from a standard curve with known cell numbers prepared in triplicate for each assay, as suggested by the manufacturers.