Anti cd27 qdot 605 clone clb 27 1

Thawed PBMCs were blocked with TruStain (BioLegend) for 5 min at room temperature, incubated with LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific) for 10 min at room temperature and stained for 30 min at 4°C with titrated concentrations of the following antibodies: (1) anti-CD19–PE-Cy7 (clone SJ25C1) and anti-CD24–APC-eFluor780 (clone SN3) (eBioscience); (2) anti-CD3–BV711 (clone OKT3), anti-CD45R/B220–BV421 (clone RA3-6B2) and anti-IgD–AF488 (clone IA6-2) (BioLegend); (3) anti-CD27–Qdot 605 (clone CLB-27/1) (Thermo Fisher Scientific); (4) anti-CD21–PE-Cy5 (clone B-ly4), anti-CD38–PE-CF594 (clone HIT2) and anti-CXCR3–PE (clone IC6/CXCR3) (BD Biosciences); and (5) anti-CD95–APC (clone DX2) (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were then washed in PBS containing 0.5% wt/vol. BSA and 2 mmol/l EDTA, fixed with 1% wt/vol. paraformaldehyde, and acquired using a modified FACSAria II flow cytometer (BD Biosciences) or an LSR Fortessa (for stimulated B cells).

Thawed PBMCs were blocked with TruStain (BioLegend, San Diego, CA, USA) for 5 min at room temperature, incubated with LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific) for 10 min at room temperature and stained for 30 min at 4°C with titrated concentrations of the following antibodies: (1) anti-CD3–APC/Fire750 (clone SK7), anti-CD8a–BV711 (clone RPA-T8), anti-CD57–PE-Cy7 (clone HNK-1), anti-CD95–PE-Cy5 (clone DX2) and anti-PD-1–BV421 (clone EH12.2H7) (BioLegend); (2) anti-CD45RA–ECD (clone 2H4LDH11LDB9) and anti-CD127–PE (clone R34.34) (Beckman Coulter, Brea, CA, USA); (3) anti-CD14–V500 (clone MSE2) and anti-CD19–V500 (clone HIB19) (BD Biosciences); (4) anti-CD4–PE-Cy5.5 (clone S3.5) and anti-CD27–Qdot 605 (clone CLB-27/1) (Thermo Fisher Scientific); and (5) anti-CXCR3–FITC (clone 49801) (R&D Systems, Minneapolis, MN, USA). Cells were then washed in PBS containing 0.5% wt/vol. BSA and 2 mmol/l EDTA, fixed with 1% wt/vol. paraformaldehyde and acquired using a modified FACSAria II flow cytometer (BD Biosciences).