Streptavidin brilliant violet 605

After washing, chunked spleen or longitudinally cut cecal tonsils were minced with the flat end of a 3-ml syringe plunger through a 40-μm cell strainer (BD Biosciences, San Jose, CA) into a 50-ml conical tube (SPL, Pocheon, Korea). To purify immune cells, red blood cells were lysed using ACK buffer (BD Biosciences) for 3 min at room temperature and then washed.
For examination of B cells and macrophages, anti-chicken MHC class II-FITC (clone 2G11), Monocyte/Macrophage-PE (clone KUL01), and Bu-1-Alexa Flour^® 647 (clone AV20) antibodies (all from Southern Biotec, Birmingham, AL) were used. To examine CD4⁺ subtypes of T cells, anti-chicken CD3-Percific Blue (clone CT-4), CD4-FITC, CD8α-SPRD (clone CT-8), TCRgd-PE (clone TCR1), CD5-biotin (clone 2-191) (all from Southern Biotec), and CD25-Alexa Fluor^® 647 (AbD Serotec) antibodies and Brilliant Violet 605 streptavidin (BioLegend, San Diego, CA) were used.
Data acquired by flow cytometry (FACS Canto II, BD Biosciences) were analyzed with FlowJo software (Tree Star, San Carlos, CA). Total cell number was determined by an automatic cell counter TC10 (Bio-Rad, Hercules, CA). The number and proportion of immune cells were calculated.

The following reagents were used for cell staining: B220-FITC (Biolegend, 103206), CD19-APC eFluor780 (eBioscience, 47-0193-82), CD21-PE (Biolegend, 123410), CD23-biotin (BD, 553137), Brilliant Violet 605 Streptavidin (Biolegend, 405229), and propidium iodide (Sigma, P4864-10ML). Cells were applied to flow cytometry using a BD LSRFortessa with FACSDiva software (BD) and analyzed using FlowJo 10.3 software (Tree Star). Follicular and marginal zone B cells (Fig. 1M) and activated lymphoblasts (Fig. 3G) were isolated using a BD Influx cell sorter. For follicular and marginal zone B-cell proportions, splenic single cell suspensions were first gated on live B220⁺ CD19⁺ lymphocytes, followed by gating on CD21/35 and CD23 (follicular B cells: CD21/35^lo CD23^hi; marginal zone B cells: CD21/35^hi CD23^lo). Cell division and proliferation indices of CTV-stained cells were determined using the cell proliferation module, and cell cycle stages were determined using the cell cycle module of FlowJo 10.3 software (Tree Star).

The sorted DC subsets and monocytes were seeded at 5 × 10⁴/well and stained with 10 μl of porcine MHC class II-DR (IgG1, clone 2E9/13), CD11R3 (CD11b-like; IgG1 clone 2F4/11), CD16 (IgG1, clone G7) mAbs (all Bio-Rad, Oxford, UK) and huCD152-muIg fusion protein (IgG2a, Enzo Life Sciences, Exeter, UK) conjugated to R-PE using Zenon^® Mouse IgG Labelling Kits (Life Technologies), and with biotinylated polyclonal anti-huCD83 antibody (R&D Systems, Abingdon, UK). Non-reactive antibodies matched by host and isotype were included at equivalent concentrations as controls. After 30 min at 4 °C the cells were washed twice with 200 μl/well of PBS/2% FBS and centrifuged as above. In the case of biotinylated CD83 antibody, streptavidin-PE (eBioscience, Hatfield, UK) was added (50 ng/well) and incubated for a further 20 min at 4 °C. All wells were washed twice and then cells were fixed by addition of 200 μl of CellFIX (BD Biosciences) and a minimum of 2.5 × 10⁴ cells were analysed on a MACSQuant Analyzer flow cytometer. To assess CADM1 expression, CD14 depleted, CD172a enriched cells were labelled as described above with the addition of anti-CADM1 mAb (clone 3E1, Caltag Medsystems, Buckingham, UK) and then detected with biotinylated anti-chicken IgY antibody (Stratech Scientific, New Market, UK) followed by streptavidin-Brilliant Violet 605 (BioLegend, London UK).