Rpmi 1640 culture medium

Neutrophils and PBMCs were isolated from human buffy coats by dextran sedimentation, erythrocyte lysis, and centrifugation in a Ficoll-Hypaque gradient (PAA Laboratories, Pasching, Austria), as previously described [13 (link)]. After isolation, the cells were suspended in RPMI 1640 culture medium (BioWest, Nuaillé, France), (Ca²⁺)-free phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA), or (Ca²⁺)-free Hanks’ balanced salt solution (HBSS; Sigma-Aldrich, St. Louis, MO, USA), depending on the test, and were stored at 4 °C until use. The buffy coats for the study were purchased from the Warsaw Regional Blood Centre. Peripheral venous blood for fractionation and preparation of buffy coats was collected in the centre from healthy male volunteers (ages 18–35 years). The donors were non-smokers, clinically recognised as healthy, and did not take any medications. The study complied with the principles of the Declaration of Helsinki. As it used a by-product material available on the market, it did not require the approval of the bioethics committee.

Monocyte adhesion to HAEC exposed to the different experimental conditions was tested in the presence and in the absence of TNF-α (5 mmol/l, 24 h). Briefly, human monocytes THP-1 were kindly donated by Dr. Magdalena Paolino (Karolinska Institute; Stockholm, Sweden) and grown in RPMI-1640 culture medium (Biowest; Nuaillé, France) supplemented with 10% FBS, 2 mol/l L-glutamine, 4,5 g/l glucose, and 1% penicillin/streptomycin, following the supplier’s recommendations. THP-1 cells were stained with 2 μmol/l of Calcein^AM fluorescent dye (Abcam) in serum-free media at 37 °C for 30 min, and washed with 10 mL of PBS. Stained cells were then resuspended in EBM2:RPMI-1640 medium (1:1), supplemented with 10% FBS, and co-cultured with HAEC under rotating conditions for 1 h at 37 °C. After incubation, non-adhering cells were removed with PBS and monolayers were fixed with 1% PFA. Co-cultures were observed under confocal microscopy and the number of adherent THP-1 cells was determined with ImageJ software.

The monoclonal antibodies for the characterization of DC and lymphocytes by flow cytometry were the following: anti-CD3 FITC, anti-CD4 APC, anti-CD8 PE, anti-CD137-biotin, anti-CD11c APC, anti-CD40-PE, anti-CD86-PE, anti-CD80-PE, anti-F4/80-Cy5 and anti-Ia/Ie-FITC from Biolegend (San Diego, CA, USA). The murine melanoma cell line B16-F10 with haplotype H-2Kb was purchased from The American Type Culture Collection, USA. RPMI-1640 culture medium was purchased from Biowest (Nuaille, France). Ge from bovine skin with a protein percentage of 78% and a water content of 8% was used. HA sodium salt from Streptococcus equi, both from SIGMA (St Louis, MO, USA) with a solubility of 5 mg/mL and an MW of ~1.5–1.8 × 10⁶ Da was used.