4.9. Neutrophil and Monocytes Transendothelial Migration Assay

Peripheral blood mononuclear cells (PBMCs) and neutrophils were separated by Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) gradient centrifugation. Human neutrophils were isolated by sedimentation of erythrocytes in 6% dextran and hypotonic lysis as previously described [26]. Monocytes were then purified from PBMCs by Percoll (GE Healthcare) gradient. Both types of cells were resuspended in RPMI 1640 supplemented with 10% FBS. Cell purity was 90% as determined by flow cytometry for both populations. Viability of cells was more than 95% in all the experiments as measured by trypan blue exclusion test.

HBMEC monolayers were established from 20,000 cells per insert on 3-μm pore size membrane Transwell plates of 6.5-mm diameter insert (Corning-Costar, Acton, MA, USA) previously treated with rat tail collagen (50 mg/mL in 1% acetic acid) (BD Biosciences) and neutralized in a saturated atmosphere of ammonium hydroxide. After 5 days, when cellular confluence was reached TEER and passive diffusion of horseradish peroxidase was measured as an indication of monolayer integrity [5]. Then, monolayers were incubated for 24 h with supernatants from B. abortus-stimulated platelets. Supernatants from platelets alone as well as non-treated HBMECs were used as negative control. Culture supernatants from Brucella-infected astrocytes and recombinant human IL-1β were used as positive control. After that, monolayers were washed and neutrophils or monocytes (1 × 10⁵ cells) were added to the upper chamber in fresh medium. Plates were incubated for 3 h at 37 °C in 5% CO₂ and transmigrated cells to the lower chamber were counted on a hemocytometer.