Anti f4 80 pe

Cells were incubated with antibody mixture including anti-F4/80-PE (BioRad), anti-Ly6G-FITC, anti-CD11b-PECy7, anti-CD45-PerCPCy5.5 and anti-Ly6C-APC or, anti-CD301-APC or anti-CD206 APC (Biolegend, San Diego, CA, USA) for 15 min and subsequently, 0.4 µl zombie dye (Biolegend) was added for 5 min. For detection of S100A9 protein intracellular staining was performed using the True-Nuclear™ Transcription Factor Buffer Set. Cells were first stained with antibody mixture including anti-CD45-PerCPCy5.5, anti-CD11b-PECy7, anti-Ly6G-APC, anti-F4/80-PE or anti-CD45-PerCPCy5.5, CD31-PE, CD90-PE-Cy7 for 15 min, washed and fixed for 45 min before intracellular staining with goat anti-S100A9 antibody (R&D) for 30 min, washing and addition of secondary antibody (anti-goat Alex 488, Life Technology) for further 30 min.
Flow cytometry was performed with BD FACSCanto^TMII or BD FACSLyric™ and data analysed with BD FACSDiva^TM Software or BD FACSuite™ Application (BD, Heidelberg, Germany). Using forward and sideward scatter single cells were gated. Living singlets were chosen from zombie-negative cells as described 31 (link).