Mouse fc receptor blocking agent

Lung and liver non parenchymal cells were harvested from out of box control and polytrauma mice. After an initial wash, cells were incubated at 37°C for 5 minutes with or without DHR123 (50 μM - Thermofischer). Mouse Fc receptor blocking agent (BD biosciences) was added to stained tubes for 10 minutes at room temperature. The cells were stained for viability (Live/Dead stain kit – Thermofisher), PE/Cy7-labeled CD68 antibody (Biolegend) and BUV395-labeled F4/80 antibody (BD Biosciences). The cells were fixed with 2% paraformaldehyde (Thermo Scientific). The assay was verified using a positive and negative control. Our positive control consisted of cells stimulated with PMA (4 μM) or LPS (100 ng/mL), and our negative control consisted of cells coming from out of box mice, not treated with any stimulant. The samples were then analyzed with a BD LSR II flow cytometer immediately after staining and results were analyzed with FlowJo version 10.7.1.

The cells were counted and equally split into different FACS sample tubes and washed by centrifugation. Mouse Fc receptor blocking agent (BD Biosciences) was added to all tubes containing cells for 5 minutes at 4°C. Cells were then stained for viability (Live/Dead stain Thermofisher) and for macrophage surface markers CD68 (Biolegend) and F4/80 (BD bioscience). Samples were kept at 4°C for 30 minutes. Samples were then washed by centrifugation and cells were fixed with 2% paraformaldehyde (Thermo Scientific). The appropriate cells were permeabilized with BD Cytofix/Cytoperm (BD bioscience) for 30 minutes. After washing the cells with the permeabilization buffer and centrifugation, antibodies that stained intracellular markers were added for 60 minutes at 4°C. Those included iNOS (Thermofisher) as a marker for the M1 phenotype and Arg1 (R&D) as markers for the M2 phenotype^{12 (link)–14 (link)}. The staining panel is included in Appendix 2. The samples were then analyzed with a BD LSR II flow cytometer, and results were analyzed with FlowJo version 10.7.1. Gating strategy is shown in Figure 1.