Preparation of cells for flow cytometry

To obtain single cells, spleen and inguinal lymph nodes were directly passed through a nylon wool sieve (70 µm). To obtain stromal cells, the spleen and inguinal lymph nodes were minced and enzymatically digested, as described³⁵. Erythrocytes (in spleen) were haemolysed (0.16 M NH₄Cl, 0.13 M EDTA and 12 mM NaHCO₃ in H₂O or ACK lysis buffer), and the cells were washed in flow cytometry buffer (2% foetal bovine serum and 2 mM EDTA in PBS), filtered through a 70 µm cell strainer and counted in an automated cell counter (Sysmex or ViCell). After Fc-blockage (anti-mouse CD16/CD32, clone 2.4G2, BD Biosciences), expression of various cell-surface markers was detected with fluorochrome-conjugated antibodies: CD19 (1D3; BD Biosciences), CD93 (AA4.1; eBioscience), IgM (Polyclonal; SouthernBiotech), CD23 (B3B4; BD Biosciences), CD21 (7G6; BD Biosciences), CD43 (S7; BD Biosciences), CD11c (N418; Biolegend), CD11b (M1/70; BD Biosciences), F4/80 (BM8; Biolegend), Gr1 (RB6-8C5; eBioscience), CD45 (30-F11; BioLegend), CD31 (390; BioLegend), podoplanin (8.1.1; BioLegend), Pdgfrα (AP5; Biolegend), rabbit anti-α1b adrenergic receptor ({"type":"entrez-protein","attrs":{"text":"EPR10336","term_id":"523375835","term_text":"EPR10336"}}EPR10336; Abcam), rabbit anti-α2b adrenergic receptor (EPR9623; Abcam), rabbit IgG monoclonal isotype control (EPR25A; Abcam), F(ab′)₂ fragment donkey anti-rabbit IgG (H + L) Alexa Fluor 647 (711-606-152; Jackson ImmunoResearch). For exclusion of nonviable cells after enzymatic digestion, LIVE/DEAD Fixable Aqua Dead Cell stain (Molecular Probes) was added.

Peritoneal cells were isolated by lavage and washed with PBS. Expression of cell-surface markers was detected with fluorochrome-conjugated antibodies after Fc-blockage (anti-mouse CD16/CD32; BD Biosciences): CD19 (1D3; BD Biosciences), IgM (Polyclonal; SouthernBiotech), CD43 (S7; BD Biosciences), and CD5 (53-7.3; BD Biosciences).

Blood was drawn from the left ventricle and prevented from clotting by storage in EDTA-coated microvette tubes (20.1288; Sarstedt). Leucocytes were enriched by haemolysis of erythrocytes and the cells were washed in flow cytometry buffer. After Fc-blockage, expression of CD19 cell-surface marker was detected with a fluorochrome-conjugated antibody, as described above.

Fluorochrome-minus-one was used as control in all flow cytometry experiments. Cells were analysed with a FACS Canto II, LSRII, FACS Aria, or Accuri C6 (all BD Biosciences); the data were further analysed with FlowJo software (Tree Star). Cells were sorted directly into Trizol (Thermofisher) after a purity check on a FACS Aria or FACS Fusion (BD Biosciences).

The gating strategies for splenic B cells (Supplementary Fig. ^8a), peritoneal B cells (Supplementary Fig. ^8b), and splenic FRCs (Supplementary Fig. ^8c) are described in the Online Supplement.