2.12. Flow Cytometry

For flow cytometric analysis, single cells were incubated with anti-CD16/CD32 (BD Biosciences) as a Fc block for 10 min at 4 °C and then stained with fluorochrome-conjugated antibodies and proper isotype controls (Table S1) for 20 min at 4 °C. Cells were washed and resuspended in PBS containing 1% bovine serum albumin (BSA) and then analyzed on a LSRII flow cytometer with FACSDiva software version 6.0 (BD Biosciences). Singlet live CD45⁺ cells were analyzed by FlowJo software version 10 (FlowJo, Ashland, OR, USA) with the following gating strategy: B cell (B220⁺CD19⁺), CD4⁺ T cell (TCRβ⁺CD3⁺CD4⁺CD8^neg), CD8⁺ T cell (TCRβ⁺CD3⁺CD4^negCD8⁺), natural killer (NK) cell (TCRβ^negNK1.1⁺), macrophage (TCRβ^negCD11b⁺CD64⁺), Neutrophil (TCRβ^negMHCII^negLy6G⁺) and dendritic cell (TCRβ^negCD64^negMHCII⁺CD11c⁺). When GFP was assessed, WT cells (GFP-negative) were stained with all antibodies used in the experiment except for the FITC/GFP channel, which is equivalent to a fluorescence-minus-one control [6].