Fa6 152

Human aortic endothelial cells (HAECs) and human THP-1 monocytic cells were used in this study. HAEC cells were purchased from Lonza (Basel, Switzerland) and cultured in endothelial growth media EBM-2 recommended by the manufacturer (Lonza, Switzerland). THP-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 (Gibco, Waltham, MA, USA). All cells were maintained at 37°C in a 5% CO₂ atmosphere. Oxidized LDL (40 μg/mL, Acesar, Waltham, MA, USA) were exposed on each cell for 24 hours to 48 hours. NQDI-1 (ASK1 inhibitor, Tocris Bioscience, Bristol, UK) was dissolved in DMSO and treated on each cell with 600 nM to inhibit ASK1 activation at 4 hours before oxLDL exposure. For the masking receptor, CD36, neutralizing the CD36 antibody (FA6-152, Abcam, Cambridge, MA, USA), was pretreated on each cell (2 μg/mL) 4 hours before oxLDL exposure. To restore ASK1, the peptide for ASK1 was synthesized to contain the active sites (Thr845) of ASK1 from amino acids 836–875.

The phagocytic function was evaluated and quantified in PBMCs by following the uptake of FITC-labeled beads (Fluoresbrite BB Carboxylate Microspheres 0.50 μm, Polysciences, Inc. Eppelheim, Germany) or the internalization of Salmonella Salp572^ФIS strain producing the green fluorescent protein (GFP-Salmonella tiphymurium) [31] (link). In detail, for the assay performed with FITC-labeled beads, cells cultivated in HEMA w/o EPO were incubated for 30 min at room temperature with the beads at 1/5 ratio (cells/beads), washed and suspended in PBS. Dead cells were excluded from the analysis by Sytox Blue staining (Molecular Probes). To study the internalization of GFP-Salmonella tiphymurium cells were incubated under shacking at 37°C with bacteria for 30 min at 1/5 ratio (cells/bacteria) followed by 2 h incubation in the presence of 100 μg/mL of gentamicin (Sigma-Aldrich). Cells were washed twice in PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich) in PBS. To evaluate the involvement of CD36 in the phagocytic process, cells were pre-incubated for 20 min at 37°C with mouse monoclonal anti-CD36 antibody (1 μg/10⁶ cells, FA6-152, Abcam Inc., Cambridge, MA, USA) and then incubated with beads or bacteria.
The percentage of phagocytic cells was evaluated in MDMs by flow cytometry comparing fluorescence intensity of cells that incorporate particles to the cell autofluorescence.