Cd23 pe cy7 b3b4

Flow cytometry was performed with the FACSAria II flow cytometer, and analysis was performed with FACSDiva 6.1.1 software (BD Biosciences) or FlowJo 8.8.4 software (TreeStar, Ashland, Ore). For spleen and BM analysis, cells were harvested, minced, and then passed through a 70-μm nylon mesh filter (BD Falcon). Erythrocytes were lysed with ammonium-chloride-potassium buffer, and the resulting cell suspension was counted and maintained in ice. Cells were stained in PBS plus 1% BSA at a concentration of 2 × 10⁶ cells per well and fixed in 1% PFA. Mouse antibodies included the following: CD11b–allophycocyanin (APC; M1/70), Ly6G–phycoerythrin (PE; 1A8), Ly6C–fluorescein isothiocyanate (HK1.4), CD19 (6D5)/CD3 (145-2C11)/NK1.1 (PK136)–Biotin, CD11c-PE (N418), PDCA-1–APC (927), Siglec H–PE (551), CD45R/B220–fluorescein isothiocyanate (RA3-6B2), CD23–PE-Cy7 (B3B4), CD21-APC (7E9), CD1d-PE (K253), and CD69-PE (H1.2-F3; BioLegend, San Diego, Calif). Dead cells were excluded by using the LIVE/DEAD Fixable Cell Stain Kit (Invitrogen). Gates were first set on live cells and singlets.

Splenocytes (SPC) were obtained from B6, Mb1 or μMT mouse recipients that were treated by the adoptive transfer of irra-OvaSPC. Cells were also collected from the draining lymph node (DLN) and non-draining lymph node (Non-DLN) and stained with the following antibodies: CD19-PB (6D5, Biolegend), IgM-PE (R6–60.2, BD Biosciences), IgD-Percp/Cy5.5 (11–26c.2a, Biolegend), CD21-APC (eBio4E3, eBioscience), CD23-PE/Cy7 (B3B4, Biolegend), CD80-PE (16–10A1, Biolegend), CD86-APC/Cy7 (GL-1, Biolegend), CD19-APC (6D5, Biolegend), Blimp1-PE (5E7, Biolegend), LAP-PE (TGF-β, TW7–16B4, Biolegend), CD365/Tim-1 PE (RMT1–4, Biolegend). Detection of antibodies was done in sera from B6 mice that were incubated with Ova⁺ SPC, and stained with CD19-PB, IgM-PE, and IgG-FITC (Poly4060, Biolegend). All results were analyzed using the FlowJo software (Treestar, Ashland, OR, USA).