Igg1 isotype control

Peripheral macrophages were isolated by lavage from the peritoneal cavity with ice-cold Mg²⁺ and Ca2+—free PBS. Cells were centrifuged at 450 x g for 10 min and resuspended in DMEM-F12/10. Cells were seeded at density of 2 x 10⁴ per well of a 24-well tissue culture plate at 37°C with 5% CO₂. Cells were further stimulated for 24 h by either CD8 (1 μg/well in 100 μl of culture medium, OX8, eBioscience) or IgG1 isotype control (1 μg/well, eBioscience). Additional subsets of cells were further stimulated for 6 h using LPS (20 ng/mL, Sigma-Aldrich). Phagocytosis assay was performed using labeled E. coli particles, according to the manufacturer’s specifications (Cytoselect, Cell Biolabs). After medium refreshment, measured amounts of E. coli particles were added to the wells and incubated for 6 h at 37°C with 5% CO₂ followed by colorimetric detection at 450 nm. In separate experiments, cells were induce the M2 phenotype [32 ] by pre-treated for 48 h with recombinant IL-4 (2 ng in 100 μL culture medium per well, Thermo-Fischer), which was followed by CD8 stimulation with phagocytosis assay performed as described above.

Purified LTA-SA was from Invivogen. Human IFNα was from PBL Assay Science. Bafilomycin A1, cytochalasin D, and dynasore were purchased from Sigma-Aldrich. Neutralizing anti-human TLR2 antibody and IgG1 isotype control were purchased from eBioscience. VIPER and CP7 peptides were from Novus Biologicals. (5Z)-Zeaenol, (5Z)-7-oxozeaenol, and the IRAK-1/4 inhibitor were purchased from Merck Millipore. BI605906, MRT67307, and MRT199665 compounds were a gift from Prof. Philip Cohen (University of Dundee) and were described previously (38 (link), 39 (link)).