Anti b220 af700 clone ra3 6b2

Single cell suspensions were prepared from spleens by mechanical homogenization between glass slides, filtered through nylon mesh, and RBC lysed with ACK lysis buffer (Thermo Fisher) for 5 minutes at 37°C. Cells were then washed and resuspended in cell sorting buffer (HBSS + 5% FBS + 5 mM EDTA). Cells were stained for 20–45 minutes at 4°C. The following antibodies/reagents were used for cell sorting experiments: anti-CD19 FITC (clone 6D5 Biolegend), anti-B220 AF700 (clone RA3–6B2 Biolegend), anti-CD95 PE-Cy7 (clone Jo.2 BD Biosciences), anti-GL7 ef450 (clone GL7 eBiosciences), NP-PE (Biosearch Technologies), anti-IgG1 APC (clone RMG1–1 Biolegend), LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Cells were washed twice and resuspended in 500µl cell sorting buffer. Bulk GC B cells (300,000 – 500,000 cells) from pooled (n = 4/group) spleens of NK cell depleted or control treated animals or from spleens of individual mice (n = 4–8/group) receiving either NK-depleting antibody or isotype control were sorted on a BD FacsAria cell sorter into 1.5 mL Eppendorf tubes containing 200 µL of cell sorting buffer.