Fluorescence activated cell sorting aria

A single-cell suspension of spleens was obtained by mechanical dissociation. Cells were suspended in flow cytometry buffer (2% FBS, 0.1% NaN₃, and 1 mM EDTA in Hanks’ balanced salt solution) at 1 × 10⁷ cells/ml and incubated with Fc block (anti-mouse CD16/32; clone 93, BioLegend) for 10 min before incubation with specific antibodies listed in table S1, except for FcγRIIB staining. Dead cells were stained out with Fixable Viability Dye (Thermo Fisher Scientific). For detection of NP-specific B cells, biotinylated NP-BSA (Biosearch Technologies) was incubated with allophycocyanin (APC)-conjugated streptavidin (BioLegend) before being added to the cell suspension. For active caspase-3 staining, Cytofix/Cytoperm buffer (BD Biosciences) was used according to the manufacturer’s instructions. For Blimp1 and TNFα staining, Foxp3/Transcription Factor Staining Buffer (Invitrogen) was used according to the manufacturer’s instructions. Cells were analyzed with a BD LSR Fortessa X-20 flow cytometer (BD Biosciences) and FlowJo software v10.7.2 (Tree Star) or sorted with a fluorescence-activated cell sorting Aria (BD Biosciences). Sorting purity of FDCs was confirmed by flow cytometry (>80%). Sorted cells then underwent stimulation or were stored in TRIzol reagent (Life Technologies) at −80°C for mRNA extraction. Some experiments were performed with frozen cells.