Cd27 bv711

Freshly isolated or ex vivo cultured human cells were washed in PBS with 5% bovine serum (FACS buffer) and subsequently stained for 30 minutes at 4°C with fluorophore conjugated antibodies against CD19, CD20, CD22, CD27, or IgD (all BD Biosciences). Cells were then washed twice in FACS buffer and analysed on a FACS Canto2 (BD Biosciences). Where appropriate dead cells were excluded using 4,6-Diamidin-2-phenylindol (DAPI; Thermo Fisher).
To analyse B cells after 9–11 days in culture, plates were spun at 400×g for 3 minutes, supernatants were taken for analysis by ELISA and cells in pellets were re-suspended and pooled for each condition. Cells were washed with PBS and stained with NIR fixable live/dead dye (Molecular probes) for 15 minutes at room temperature and washed with FACS buffer (2%BSA, 2mMEDTA, PBS). Live/dead staining was followed by an incubation with Fc Block reagent (BioLegend) for 10 minutes at RT and finally staining with a mix of labelled antibodies: CD45-BV785, CD19-FITC, CD27-BV711, CD38-APC, CD138-PE, and IgD-BV421 (all BioLegend) following the manufacturers recommendations for 30 minutes at 4C. Stained cells were washed and fixed with 2% PFA in PBS for 10 minutes at RT. Stained cells were analysed in a Beckton Dickinson Fortessa with 355, 405, 488, 561, and 633nm lasers.

B cells were incubated with an antibody cocktail containing L/D marker (eBioscience Fixable Viability Dye eFluor 780) and antibodies CD10 BUV737 (BD #612826), CD20 PECy7 (BioLegend #302312) and CD27 BV711 (BioLegend #302834) and IgD BUV395 (BD #563813) for 30 min at 4°C. Labelled B cells were sorted into naïve (CD10⁻CD20⁺CD27⁻IgD⁺) and memory (CD10⁻CD20⁺CD27⁺ and CD10⁻CD20⁺CD27⁻IgD⁻) B cells using the FACSAria™ Fusion sorter (BD Biosciences) (gating strategy shown in Figure S1A).

All flow cytometry data were acquired on BD LSRII flow cytometer, and results were analyzed using FlowJo software (v.X.0.7, Tree Star, Ashland, OR). Compensation was carried out using single-color controls of either cells or beads (OneComp eBeads, eBioscience). Appropriate isotype controls, fluorescence minus one (FMO), or NTD cells were used to validate gating. All samples were stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies) or Fixable Viability Dye eFluor 780 (eBioscience) before antibody staining. The following fluorochrome-conjugated mouse anti-human antibodies were used: CD3 PerCP-Cy5.5 (BioLegend, clone UCHT1), Vδ2 PE (BioLegend, clone B6), Vδ2 fluorescein isothiocyanate (FITC) (Miltenyi, clone 123R3), Vδ1 APC-Vio770 (Miltenyi, clone REA173), Vδ1 PE (Miltenyi, clone REA173), anti-TCR γδ PE-Vio770 (Miltenyi, clone 11F2), anti-TCR αβ Brilliant Violet (BV) 421 or PE-Cy7 (both BioLegend, clone IP26), QBend10 APC (R&D Systems, clone 4H11), CD27 BV711 (BioLegend, clone O323), CD45RA PE-Cy7 (BioLegend, clone H100), PD1 FITC (BioLegend, clone EH12.2H7), TIM3 BV605 (BioLegend, clone F38-2E2), and TCR Vβ12 FITC (Abcam, clone S511).