Anti cd3 clone 1f4

Single cell suspensions of splenocytes, bone marrow, and lymph node cells were stained for B and T cells and analyzed by flow cytometry (BD LSR II at the UWCCC Flow Cytometry Laboratory) using FlowJo software (TreeStar) as previously described.^{15 (link)} B cell populations were defined as: transitional B cells IgD⁺ CD45R⁺ CD24^+(hi) CD38^+(hi); splenic marginal zone B cells CD45R⁺ HIS57⁺ CD45RA⁺; and lymph node naïve B cells (IgD⁺ CD45R⁺ CD27⁻). Antibodies used for B cell stains included: anti-IgD (clone MARD-3, BioRad); anti-CD45R (clone HIS24, eBioscience); anti-CD24 (clone REA405, Miltenyi Biotec); anti-IgM (clone G53-238, BD Pharmingen); anti-CD3 (clone 1F4, BD Horizon); anti-CD27 (clone LG.3A10, BD Horizon); and anti-CD38 (clone 14.27, BioLegend). Antibodies for marginal zone B cell staining included: HIS57 (BD Pharmingen) and anti-CD45RA (clone Ox33, BD Pharmingen). Antibodies for T cell staining included: anti-CD8 (cloneOX-8, BioLegend); anti-CD45R (clone HIS24, eBioscience); anti-CD4 (clone W3/25, Biolegend); anti-CD3 (clone 1F4, BioLegend); anti-CD25 (clone OX-39, BioLegend); anti-FoxP3 (clone 150D, BioLegend); anti-CD278 (clone C398.4A, BioLegend); and anti-CXCR5 (clone EPR8837, Abcam). Surface Ig staining was detected using: anti-IgG (Jackson ImmunoResearch); anti-IgM (clone G53-238, BD Pharmingen); and anti-IgD (clone MARD-3, BioRad).

Single cell suspension of splenocytes were stained for B cells, T cells, and macrophages and analyzed by flow cytometry (BD LSR II) using FlowJo software (TreeStar, version 10.5.3), as previously described.^{32 (link)} B cell subsets were identified as transitional B cells (CD3⁻IgD⁺CD45R⁺CD38⁺CD24⁺⁺) or naïve B cells (CD3⁻IgD⁺CD45R⁺CD27⁻). T cell subsets were identified as T helper cells (CD3⁺CD4⁺), cytotoxic T cells (CD3⁺CD8⁺), T regulatory cells (CD3⁺CD4⁺CD25⁺FoxP3⁺), or T follicular helper cells (CD3⁺CD4⁺CD278⁺CXCR5⁺). Antibodies for flow staining included: anti-CD3 (clone 1F4, BioLegend), anti-IgD (close MARD3, BioRad), anti-CD45R (B220) (clone HIS24 eBioscience), anti-CD38 (clone 14.27 BioLegend), anti-CD24 (clone ML5, BD Horizon), anti-CD27 (clone LG.3A10, BD Horizon), anti-CD4 (clone W3/25, Biolegend), anti-CD8 (cloneOX-8, BioLegend), anti-CD278 (clone C398.4A, BioLegend), anti-CXCR5 (clone EPR8837, Abcam), anti-CD25 (clone OX-39, BioLegend), and anti-FoxP3 (clone 150D, BioLegend). Ghost Dye Red 780 Viability Dye was used in all experiments.