Cd27 af700

2 × 10⁶ PBMCs were stained with fixable blue dead cell stain (ThermoFisher) or Zombie NIR™ Fixable Viability Kit (Biolegend) in PBS, followed by washes and surface marker staining in Brilliant™ Stain Buffer (BD) with antibodies (Biolegend unless stated): CD3-BV786 (317330), CD4-BUV395 (BD) (563550), CD8-BV421 (301036), CD27-AF700 (356416), CD45RA-PE-Cy7 (304126), CD25-PEdazzle594 (356126), CD127-BV711 (351328), iTCR-PE (342904), CD19-AF488 (302219), IgD-BV510 (348220), HLA-DR-BV786 (307642), CD38-BV421 (356618), CD14-BV711 (301838), CD16-PEdazzle594 (302054), CD303-PerCP-Cy5.5 (354210) followed by subsequent washes and fixation in 2% PFA. Stains and washes were carried out in Biolegend cell staining buffer. Data was acquired using a BD LSRFORTESSA X-20 flow cytometer (1-2 × 10⁶ cells per sample) and analysis performed using FlowJo Single Cell Analysis Software (TreeStar). Cytometer Setup and Tracking (CS&T) (BD) beads were used to monitor cytometer performance. Application settings were applied prior to compensation to ensure that all immunophenotyping data was comparable over time. Gating strategies are shown in Supplementary Figure 1.

Cells were stained with the following antibodies: FcRL4-APC (Biolegend), IL-6-PE, CD23-PE-Cy7, CD19-PE-Texas Red, CD10-Pe-Cy5, CD21-V450, CD27-AF700, phospho-p38-PE, phospho-Erk-AF647, phospho-p65-PE, phospho-C-Jun-FITC, (BD Biosciences), Sheep anti-rabbit IgG-DyLight 488 (Biolegend), purified hematopoietic cell kinase (Hck), and phospho-Hck (Abcam).