Cd21 apc b ly4

Cells were resuspended at a concentration of 10⁶ cells/100 μl fluorescence-activated cell sorting (FACS) buffer (1X PBS, 2 mM EDTA, 1% BSA). Cells were stained with Fc block (BD Biosciences) for fifteen minutes, stained with antibody-fluorophores for one hour on ice, then washed with ten volumes of FACS buffer. The following antibodies were used: B220-PE-Cy7 (RA3–6B2, Tonbo), CD11b-APC-Cy7 (M1/70, Tonbo), CD45.1-FITC (A20, Tonbo), CD45.2-PerCP-Cy5.5 (104, Tonbo), CD90.2-APC-Cy7 (30-H12, BioLegend), F4/80-APC-Cy7 (BM8, BioLegend), CD138-BV711 (281–2, BD), CD21-APC (B-ly4, BD), CD43-PE (S7, BD), GL7-eFluor660 (GL-7, eBioscience), CD23-eFluor450 (B3B4, eBioscience), IgM-FITC (II/41, eBioscience), Annexin V-APC (kit number 88–8007-72, eBioscience), and Zombie Yellow Fixable Viability Dye (kit number 423104, BioLegend). An LSRII was used for analysis and an ARIAII was used for sorting (BD Biosciences). All flow cytometry data was analyzed with FlowJo version 9.9.5. Preceding all flow cytometry analyses presented is the following gating strategy: 1) lymphocytes (forward scatter [FSC]-area by side scatter [SSC]-area), 2) singlets (FSC-width by FSC-height), 3) singlets (SSC-width by SSC-height), 4) live cells (viability dye⁻), 5) exclusion of non-B cell lineage cells (Thy1.1⁻F4/80⁻CD11b⁻).