BMDMs were stimulated in RPMI-10 with 0.1 or 1.0 μg per plate of plate-coated MA or TDM in the presence of 10 ng/ml of recombinant IFN-γ for 24 h. For the assessment of TNF involvement in NO production, carrier-free recombinant mouse TNF (Biolegend #575202), LEAF purified anti-mouse TNF-α antibody (MP6-XT22; BioLegend #506331), or LEAF purified Rat IgG1 κ Isotype Ctrl antibody (BioLegend #400457) were added to the culture. The culture supernatants were collected and mixed with Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, and 2.5% phosphoric acid) at a 1:1 ratio, reacted for 10 min, and the absorbance at 550 nm was measured by a VERSAMax microplate reader using SoftMaxPro software. The nitrite concentration was calculated according to the standard curve.

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