mAbs against the following mouse antigens were used: Alexa Fluor 488-conjugated anti-CD45, allophycocyanin (APC)-conjugated anti-Ly6G, Pacific Blue-conjugated anti-CD11b, peridinin-chlorophyll-conjugated anti-Ly6C, phycoerythrin-cyanine 7-conjugated anti-CD206 (all from BioLegend, San Diego, CA, USA), and APC-conjugated anti-CD204 (R&D Systems, Minneapolis, MN, USA). To distinguish dead cells from live cells, the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used.
The single-cell suspensions obtained as described above were stained for 20 minutes at 4 °C using the indicated mAbs at predetermined optimal concentrations for six-color immunofluorescence analysis. Stained samples were analyzed using the FACSCanto II system (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software version 7 (FlowJo, Ashland, OR, USA).
The single-cell suspensions obtained as described above were stained for 20 minutes at 4 °C using the indicated mAbs at predetermined optimal concentrations for six-color immunofluorescence analysis. Stained samples were analyzed using the FACSCanto II system (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software version 7 (FlowJo, Ashland, OR, USA).