Sorting strategy of different B-cell subpopulations starting from CD19⁺-enriched B cells. CD19⁺-enriched B cells were stained with CD19-PE, CD3-Pe-Vio770, CD20-APC-eFluor780, CD43-FITC, CD27-APC, and CD69-PeCy5. (A) Doublets were excluded based on forward scatter (FSC)-H and FSC-A profile, cells with lymphocyte morphology were gated on FSC-A vs SSC-A profile, T cells were excluded by gating CD3⁻ cells. Naive B cells (nai) are defined as CD3⁻CD19⁺CD20⁺CD27⁻CD43⁻, memory B cells (mem) as CD3⁻CD19⁺CD20⁺CD27⁺CD43⁻, the proposed B-1 cells as CD3⁻CD19⁺CD20⁺CD27⁺CD43⁺CD69⁻. (B) Plasmablasts (PB) were gated as CD3⁻CD19⁺CD20⁻CD27⁺CD43⁺ with the plot showing CD3⁻CD19⁺CD20⁻ cells as gated on the lower left plot of (A).