Isolated immune cells were stained with the Live/Dead Fixable Blue Dead Cell Stain Kit from Life Technologies (Grand Island, NY, USA) to exclude dead cells. Anti-mouse CD16/CD32 mAb from BD Biosciences (San Jose, CA, USA) was used to block non-specific Fc receptor binding and incubated for 20 min at 4 °C. Cells were immunostained for 30 min at 4 °C with surface antibodies (Table 1), fixed and permeabilized with BD Biosciences Cytofix Cytoperm Plus according to the manufacturer’s protocol, and stained for intracellular CD68 for 30 min at 4 °C (Table 1). Data were acquired on a BD Biosciences LSR II instrument and analyzed using the FlowJo v10.1 software (Ashland, OR, USA). Table 1Flow cytometry and immunofluorescence antibodiesAntigenColorCloneCompanyCat. no.CD11bAPCM1/70Biolegend101212CD45PE/Cy730-F11eBioscience25-0451-82CD68PerCP/Cy5.5FA-11Biolegend137010CD86FITCMHC-IIAPC/Cy7M5/114.15.2Biolegend107628CD206PEC068C2Biolegend141706CD163 (goat host)n/aK-18Santa Cruzsc-18796Goat IgGQdot 525n/aInvitrogenQ22072Primary antigenPrimary dilutionCompanyCat. no.Secondary and fluorSecondary dilutionIBA11:500WakoAnti-Rb-4881:500CD681:500AbD SerotecMCA1957Anti-Rt-6471:500Secondary antibodies from Life Technologies
