Mcp 1

The hCMEC/D3 cells were seeded at a density of 40–50,000 cells/cm² in hanging cell culture inserts (PET, 5-µm pores, Millipore) pre-coated with rat collagen type 1 (Sigma). The cells were kept in culture for 7 days until ready for the transmigration and permeability assays. Infected monocytes were extensively washed in RPMI 2% FBS media before addition to the transwell inserts containing the hCMEC/D3 cells. In parallel, the media in the bottom chamber was replaced with fresh media containing 200 ng/mL of MCP-1 (Miltenyi Biotech). Transmigration was allowed to occur overnight (about 17 h). Monocytes were recovered from the top and bottom chambers of the insert and prepared for flow cytometry analysis.

To analyze chemotaxis, a specific 96‐well cell migration system (Neuro Probe) was applied as previously described.^[54] Briefly, the chemotactic stimulus was added in a volume of 37.5 µL per well. A membrane filter with 5 µm pores was placed onto the plate. 3 × 10⁴ cells in 40 µL diet medium consisting of VLE‐RPMI (Biochrom) and 0.1% autologous serum were added on the membrane. Chemotaxis was induced by 50 or 100 µg mL⁻¹ NID1 or without stimulus (negative control). MCP‐1 (50 ng mL⁻¹, Miltenyi Biotec) served as positive control. After incubation for 3 h at 37 °C in a 5% CO₂ incubator, the membrane was carefully removed. Monocytes that adhered to the membrane were fixed with methanol (Merck) and labeled with Hemacolor staining kit (Merck). After microscopic documentation using ProgRes CapturePro 2.8.8 (Jenoptik), the number of migrated monocytes was determined using ImageJ Version 1.4.3.67 (National Institutes of Health). The number of migrated cells was normalized to the negative control.

All transmigration assays were performed by employment of Millicell Cell Culture Inserts (Millipore, 8 µM pore size, 12 mm diameter). Primary monocytes or THP-1 cells were incubated in RPMI medium containing 1 µM calcein-AM at 37 °C for 60 min. The cells were washed with RPMI twice. 3×10⁵ primary monocytes or 5×10⁵ calcein-labelled THP-1 cells were re-suspended in 400 µl RPMI medium and seeded into upper transmigration chamber. Depending on the experiment, the cells transmigrated through uncoated or HUVEC monolayers coated membranes towards lower chamber filled with 600 µl RPMI medium supplemented with 50 ng/ml MCP-1 (Miltenyi) or 10% NS or HD or PD or CKD5 sera. In some experiments, THP-1 cells transmigrated towards HUVEC monolayers created on the bottom of lower chamber in the presence of RPMI only in both chambers. All transmigrations were performed at 37 °C for 60 min in the absence of FCS. Transmigrated cells were visualized by fluorescence microscopy (Biozero BZ-9000, Keyence) and counted in 10 microscopic fields for each situation by employment of ImageJ software (Wayne Rasband, National Institutes of Health, USA). In some experiments transmigrated cells were counted by FACS. Experiment was repeated at least three times.