Monocyte Migration Assay Protocol

Human monocytes were isolated as described previously(18 (link), 20 , 29 (link)). Briefly, monocytes were isolated by centrifugation of citrate-phosphate-dextrose-adenine (CPDA)-coagulated blood (S-Monovettes, Sarstedt, Nümbrecht, Germany) on a Ficoll-Paque gradient (GE Healthcare). Leukocytes were cultured overnight at 37°C, 5% CO₂ in a cell culture flask in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FCS (Gibco) and 1% Penicillin/Streptomycin (Sigma-Aldrich, St. Louis, MO, USA). For the migration assay, non-adherent leukocytes were removed and adherent cells were trypsinised (Gibco), centrifuged, and resuspended in medium. Monocyte migration was performed in a modified 48-well Boyden chamber (NeuroProbe Inc., Geithersburg, MD, USA), using a 5 μm pore filter to separate the 2 compartments. Monocytes were added in the upper compartment and 20 μg/ml S100A9 (abcam) or 200 nmol/L CyPA (R&D Systems) or CyPA together with 20 μg/ml of the potential antibodies or _rIgG_2a (Invitrogen, Carlsbad, CA, USA) were added in the lower compartment. The receptor blocking antibody αCD147 and _mIgG₁ were added to the monocytes into the upper compartment. After 4 hours at 37°C, 5% CO₂ the filter was fixed in Methanol, stained in a May-Grünwald/ Giemsa solution (Merck Millipore, Darmstadt, Germany) and the migrated cells on the filter were counted using a microscope.