Splenic B cells from young (5 weeks) or aged (12 months) Adig mice were magnetically enriched using a mouse B cell Isolation Kit with a QuadroMACS separator and LS columns according to the manufacturer’s instructions. Enriched B cells were suspended in DAPI in FACS buffer and sorted on Aire-GFP− viable singlets. Subsequently, 2 × 105 sorted B cells were cultured for 3 days with or without agonistic anti-CD40 monoclonal antibody (FGK45; Bio X Cell) (10 μg/mL) and recombinant mouse IL-4 (PeproTech) (5 ng/mL) in flat-bottom 96-well plates. For qRT-PCR, RNA extraction, cDNA synthesis, and qPCR were performed on 2 × 105 cells using Taqman Gene Expression Probes (Thermo Fisher Scientific) for Aire and Hprt and gene expression values normalized as previously described.
For flow cytometry analysis, 6 × 105 to 1 × 106 stimulated B cells were stained with CD19-A594 (6D5; BioLegend) and suspended in DAPI in FACS buffer. Aire-GFP expression was analyzed on CD19+ viable singlets using a BD LSRII flow cytometer with BD FACSDiva and FlowJo® flow cytometry analysis software.
For flow cytometry analysis, 6 × 105 to 1 × 106 stimulated B cells were stained with CD19-A594 (6D5; BioLegend) and suspended in DAPI in FACS buffer. Aire-GFP expression was analyzed on CD19+ viable singlets using a BD LSRII flow cytometer with BD FACSDiva and FlowJo® flow cytometry analysis software.