Multiparametric Flow Cytometry Analysis of B Cell Subsets

Spleen cell suspensions or cultured B-cells were stained with the following antibodies as described:^{25 (link),46 (link)} anti-CD138-PE (clone: 281-2); anti-CD95-PE (clone: Jo2); IgM-APC (clone II/41); anti-IgD-PE (clone 11-26c.2a); and anti-CD23-PE (clone B3B4) (all BD Pharmingen); and anti-B220-PerCP (clone: RA3-6B2); anti-CD21-APC (clone: 7E9); anti-CD24 (HSA)-PE (clone: 30-F1), anti-CD93-PE (clone: AA4.1); and anti-CD23-Pacific Blue (clone: B3B4) (all Biolegend); and anti-CD19-CF594 (clone: 1D3) (BD Horizon); and PNA-Biotin (Vector Laboratories) followed by Streptavidin-APC (BD Pharmingen). Annexin V/7-AAD stainings were performed using the APC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend). For DNA content analysis, cells were lysed and stained with propidium iodide (PI). The cells were analyzed on a FACSCalibur or a LSRII (Becton Dickinson). Transitional B-cells were identified by gating on B220⁺CD93⁺ lymphocytes.^{39 (link)} GC B-cells were identified by gating on B220⁺ lymphocytes. eGFP⁺ and eGFP⁻ CD138^hi plasma cells were identified through the lymphocyte gate. Data were analyzed using FlowJo software.