Lta from staphylococcus aureus

We performed a natural antigen processing assay (NAPA) as described previously (13 (link)). Briefly, immature mo-DCs were seeded in 6-well plates at a density of 1 × 10⁶ cells in 1 mL of RPMI 1640 and 10% fetal bovine serum, supplemented with 2 mM L-glutamine and 20 μM ß-mercaptoethanol in duplicate. Immature mo-DCs were left at rest or stimulated with 1 μg/mL purified lipoteichoic acid (LTA) from Staphylococcus aureus (InvivoGen, San Diego, CA) or live B. burgdorferi strains A3 or B31 at multiplicities of infection of 1 or 10 bacteria per cell for 8 and 24 h. Cells were incubated at 37°C, 5% CO₂, and >95% humidity. At the end of the incubation period, all samples were harvested into a microcentrifuge tube, pelleted at 1,200 rpm at room temperature for 3 min. Cells were washed with PBS/EDTA (PE) Buffer and pelleted at 1,200 rpm at room temperature for 3 min. Cells were stained with CD14-PE (BD Pharmingen), CD11c-PerCP (BioLegend), HLA-DR-BV510 (BD Horizon), and Blue Fluorescent Reactive Dye (Life Technologies) for 15 min in BV Buffer (BD Horizon). At the end of the incubation period, cells were washed twice in PBS and resuspended in 100 μL PBS for flow cytometry analysis in a BD FACSAria II flow cytometer (BD Biosciences). All data was gated on CD14⁻/ CD11c⁺ monocyte derived dendritic cells.

Cell viability reagent WST-1 was purchased from Roche Diagnostics, Almere, The Netherlands. Human serum and human plasma purified lipoproteins (VLDL, LDL, HDL) and apolipoproteins (ApoA, ApoB, ApoC1, ApoC2, ApoC3) were all purchased from Sigma-Aldrich. Human lipoprotein deficient serum was from Merck, Amsterdam, the Netherlands. Heat-killed Staphylococcus aureus (HKSA), Escherichia coli (HKEB), Salmonella typhimurium (HKST) were all purchased from Invivogen, Toulouse, France. Ultrapure LPS derived from E. coli K12 and purified LTA from Staphylococcus aureus (both from Invivogen, Toulouse, France) were used at the indicated concentrations. Synthetic bacterial lipopeptides Pam₃CSK₄, Pam₂CSK₄, FSL-1 (all from EMC microcollections, Tübingen, Germany) were used at the indicated concentrations. Non-phagocytic HEK293 TLR2-TLR6, HEK293 TLR4 stable transfectants and HEK293 TLR null control cells were purchased from Invivogen, Toulouse, France. HEK293 TLR2-TLR6 and HEK293 TLR null transfectants were stably transfected with the NFκB reporter plasmid pNiFty2-Luc, HEK293 TLR4 cells contained the NFκB reporter pNiFty2-SEAP (Invivogen, Toulouse, France). Cells were maintained in DMEM (Invitrogen) supplemented 10 % FBS, 4.5 g/L glucose and the appropriate antibiotics according to the manufacturer’s protocols.