Cd27 biotin

To enrich naive B cells by negative selection, tonsil mononuclear cells were thawed, washed, counted, resuspended in MACS buffer (PBS + 0.5% BSA + 2 mM EDTA) and labeled for 10 min on ice with B cell isolation kit II antibody cocktail (Miltenyi) supplemented with the following additional antibodies: CD10-biotin (Miltenyi), CD27-biotin (Biolegend), CD77-FITC (Biolegend). Cells were then washed, labeled with anti-biotin and anti-FITC microbeads (Miltenyi), and incubated on ice for 20 min. After washing, the cells were resuspended in MACS buffer, loaded onto one or more LS columns (Miltenyi) placed in MACS separation magnets (Miltenyi), and the flow-through (unlabeled fraction) was collected. Post-sort B cell purity was confirmed on a FACS Canto I using the flow cytometry staining procedures described above, and were at least 85% pure, but typically >90%. Where indicated, to obtain a GC-depleted fraction, sorts were performed as above, except that CD27 antibody was omitted.

The following antibodies were purchased from Biolegend: CD3 BV510, CD49b (DX5) APC-Cy7, NKp46 BV421, CD11b PE, Ly49D PE, Streptavidin BV650, CD27 biotin, KLRG1 BV605, KLRG1 PerCP/Cy5.5, CD69 PerCP/Cy5.5, and Ki67 APC. The following antibodies were purchased from eBioscience: CD107a PerCP-eFluor710, Ly49I PE, CD94 FITC, IFNγ APC, and Ly49H PE-Cyanine7. The following antibodies were purchased from BD Biosciences: NKG2A/C/E BV605. The following antibodies were purchased from Miltenyl Biotec: NKG2D (CD314) biotin, Ly49D biotin, and Ly49D PerCP-Vio700. To determine viability Invitrogen Fixable live/dead Aqua stain was used.