Anti cd16 cd32 antibody clone 93

Single-cell suspensions of spleen tissue were prepared in complete DMEM as described above: 1 × 10⁶ cells were pre-incubated with 1 μg of Fc receptor block (anti-CD16/CD32 antibody clone 93; 14–0161-81, eBioscience, San Diego, CA), prior to staining with 0.1 μg of antibodies for 15 min on ice. Rat anti-mouse CD19 fluorescein isothiocyanate (anti CD19-FITC) antibody clone 1D3 (553785), anti-mouse CD4-phycoerytherin (anti CD4-PE) antibody clone GK1.5 (553730), and anti-mouse CD8-phycoerytherin-cyanine 5 (anti CD8-PE-Cy5) antibody clone RPA-T8 (555368), all purchased from BD Biosciences (San Jose, CA, USA) were used. The stained cells were analyzed using Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA) and FlowJo software (Tree Star Inc., Ashland, OR, USA).

Depending on the panel, dead cell discrimination with a fixable viability dye was performed prior to surface marker staining. After washing in PBS, a maximum of 1 × 10⁶ cells was stained with freshly reconstituted fixable viability dye at 1 µL/1 mL PBS for 30 min. Excess dye was removed by washing in PBS. Unspecific binding to Fc-receptors was blocked by resuspending in anti-CD16/CD32 antibody (clone 93, ebioscience, 1:100). After 5–10 min incubation appropriate antibody cocktails for surface marker staining (respective panels in Online Resource data) were added 2× concentrated and cells were stained for 20min on ice.
Prior to acquisition, cells were passed through a 37 µm filter and viability marker 7-AAD (Becton Dickinson, Erembodegem, Belgium) was added at 3 µL/1 × 10⁶ cells if required. Samples were analyzed either at a BD FACSCanto II or a BD LSRFortessa (BD Biosciences, San Jose, CA, USA). Cell sorting of neutrophils and macrophages was performed at a BD FACSAria1 machine (BD Biosciences) and cells were sorted into FACS buffer. Data were analyzed using FlowJo v10 (Tree Star, Inc., Ashland, OR, USA).