Assay of Macrophage Responses to LPGs

Thioglycollate-elicited peritoneal macrophages were extracted from C57BL/6 and C57BL/6 (TLR2 and TLR4 knockouts) as previously reported (Ibraim et al., 2013 (link); Nogueira et al., 2016 (link)). Briefly, recovered cells (3 × 10⁵ cells/well) were washed with fresh RPMI and cultured in the same medium supplemented with 2 mM glutamine, 50 U/ml of penicillin and 50 μg/mL streptomycin, 10% Fetal Bovine Serum in 96-well culture plates (37°C, 5% CO₂). Cells were primed with gamma interferon (IFN-γ) (3 IU/mL) for 18 h prior to incubation with LPGs (10 μg/mL) from all strains and controls for 48 h. Those included LPS (100 ng/mL, positive) and medium (negative). The nitrite concentration was measured by Griess reaction. For cytokine detection, supernatants were collected and IL-1β, IL-6, IL-12, and TNF-α were determined using BD CBA Mouse Cytokine assay kits according to the manufacturer's specifications (BD Biosciences, CA, USA). Flow cytometry measurements were performed on a FACS Calibur flow cytometry (BD Bioscience, Mountain View, CA, USA). Cell-Quest TM software package provided by the manufacturer was used for data acquisition and the FlowJo software 7.6.4 (Tree Star Inc., Ashland, OR, USA) was used for data analysis (Nogueira et al., 2016 (link)).