Anti igg3 pe cy7

For ex vivo analysis of B‐1 cells, 1 × 10⁶ single cells from PL, BM, or spleen were incubated with anti‐mouse CD16/32 (Biolegend) for 15 min on ice for blocking of unspecific Fc‐binding sites. Subsequently, cells were stained with anti‐CD19 FITC, anti‐CD43 PE‐Cy7, anti‐CD5 APC, anti‐IgM APC‐Cy7, and anti‐CD138 PE antibodies (Biolegend and BD Bioscience) for 15 min on ice. To assess cell death, cells were incubated with 7‐aminoactinomycin D (BD Bioscience) before analysis for at least 15 min, but no longer than 60 min.
To analyze proliferation and surface immune globulins after in vitro stimulation, CFSE‐stained cells were incubated with anti‐CD19 V450, anti‐CD43 PE‐Cy7, anti‐CD5 APC, anti‐IgM APC‐Cy7, anti‐IgD‐PerCP, and anti‐IgG₁ PE (LPS/IL‐4) or with anti‐CD19 APC‐Cy7, anti‐CD43 PE, anti‐CD5 APC, anti‐IgA BV421, anti IgG_2ab BB700, and anti‐IgG₃ PE‐Cy7 (LPS only) (Biolegend and BD Bioscience).
All measurements were performed at the BD FACS Canto II and biaxial gating was done with FlowJO Version 10.

For flow cytometry, 1 × 10⁶ single cells from PL, BM, and spleen were incubated with anti‐mouse CD16/32 (BioLegend) for 15 min to block unspecific Fc‐binding sites. Subsequently, cells were fluorescently stained in two mixes to identify B‐1 cells, CD138⁺ cells, splenic B‐2 cells, and BM subpopulations: B220‐FITC, anti‐CD5‐PerCP‐Cy5.5, anti‐IgD‐V450, anti‐IgM‐APC‐Cy7, anti‐CD43‐PE, anti‐CD138‐APC and anti‐CD23‐BV510, anti‐B220‐PerCP, anti‐CD21‐FITC, anti‐CD23‐BV510, and GLY7‐PE (BioLegend and BD Bioscience). For identification of PBs and plasma cells (PCs), fluorescent staining was performed using anti‐B220‐FITC, anti‐CD19‐V450, anti‐TACI‐APC, anti‐CD138‐PE, and 7‐AAD for exclusion of dead cells.
To analyze surface immune globulins after in vitro stimulation, splenic B cells were incubated with anti‐CD19‐V450, anti‐CD43‐PE‐Cy7, anti‐CD5‐APC, anti‐IgM‐APC‐Cy7, anti‐IgD‐PerCP and anti‐IgG₁‐PE (LPS/IL‐4) or with anti‐CD19‐APC‐Cy7, anti‐CD43‐PE, anti‐CD5‐APC, anti‐IgA‐BV421, anti‐IgG_2ab‐BB700, and anti‐IgG₃‐PE‐Cy7 (LPS only) (Biolegend and BD Bioscience). Proliferation was analyzed via carboxyfluorescein succinimidyl ester‐staining.
All measurements were performed at the BD FACS Canto II and biaxial gating was done with FlowJo Version 10. For flow cytometry gating strategies the reader is referred to Figure S2.