Normal murine igg antibodies

Splenocytes (2 × 10⁶ cells) were incubated with either monoclonal antibody (mAb) against CD16/CD32 (clone 2.4G2, Thermo Fisher Scientific, Waltham, MA, USA), or normal murine IgG antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for treatment of IgG receptors on the cell surface, and stained with allophycocyanin (APC) anti-mouse F4/80 mAb (clone QA17A29) and phycoerythrin (PE) anti-mouse/human CD11b mAb (clone M1/70) together with 7-AAD viability staining solution. Monoclonal Abs and 7-AAD were from BioLegend. Fluorescence intensity of the cells in 7-AAD negative population (viable cells) was measured by FACSLyric (BD Bioscience, Franklin Lakes, NJ, USA).

To detect the expression levels of co-stimulatory molecules on the cell surface of RAW-267.4 cells and BMDCs, fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD40 (clone 3/23, Biolegend, San Diego, CA, USA), CD80 (clone 16-10A1, Biolegend, San Diego, CA, USA), CD86 (clone GL-1, Biolegend, San Diego, CA, USA), or MHC class II (I-A/I-E) mAbs (clone M5/114.15.2, Biolegend, San Diego, CA, USA) were used. In all stainings, IgG receptors on the cell surface were blocked using either monoclonal antibody (mAb) against CD16/CD32 (clone 2.4G2, Thermo Fisher Scientific, Waltham, MA, USA), or normal murine IgG antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). BMDCs were additionally stained with allophycocyanin (APC)-conjugated anti-mouse CD11c mAb (clone N418, Biolegend, San Diego, CA, USA) to gate the DC population. Fluorescence intensity of stained cells (10,000 cell detection) was measured with flow cytometry using a FACSLyric (BD Bioscience, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo V. 7 (BD Bioscience, Franklin Lakes, NJ, USA).