2.4. Assessment of Macrophage Activation Markers

Expression of surface activation markers on BMDMs was quantified by flow cytometric analysis following staining with specific antibodies conjugated with fluorescent molecules. Aforementioned BMDMs were rinsed with cold PBS containing 2% FBS and incubated with mouse CD16/CD32-specific mAbs (Fc block, eBioscience) for 15 min at 4 °C to inhibit non-specific bindings. Cells were then stained with fluorescence-conjugated antibodies as follows: fluorescein isothiocyanate-conjugated anti-mouse F4/80 (F4/80-FITC, eBioscience), anti-mouse CD11b conjugated with peridinin-chlorophyll protein-cyanin5.5 (CD11b-PerCP-Cy5.5, eBioscience), allophycocyanin-conjugated anti-mouse CD11c (CD11c-APC, eBioscience), and phycoerythrin-conjugated anti-mouse CD206 (CD206-PE, eBioscience) for 10 min at 4 °C, simultaneously. Cells were then washed twice and resuspended in ice-cold PBS followed by analysis using a flow cytometer (Accuri™ C6, BD Bioscience, San Jose, CA, USA). The double positive BMDMs for F4/80 and CD11b were gated as mature macrophages. Relative expression of activation markers was determined by mean fluorescence intensity (MFI) and normalized to the MFI of the normal died-fed control using FlowJo^® software (BD Bioscience).