Anti cd27 bv605

After stimulation, PBMCs were labeled with viability marker LIVE/DEAD Near-IR fluorescent reactive dye (Thermo Fisher) for 30 min and subsequently stained for 20 min with the following surface markers: anti-CD3-PerCP (BioLegend), anti-CD4-BV786, anti-CD8-BV510, anti-CD27-BV605, anti-CD38-PE, and anti-HLA-DR-BV421 (BD Bioscience). Cells were then fixed and permeabilized with the Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher) and stained for 30 min with intracellular markers: anti-IFNγ-APC, anti-TNFα-PE-Cy7 and anti-Ki-67-FITC (BD Bioscience). The complete list of antibodies, conjugated proteins and dilutions can be found in Supplementary Table 1. All incubation processes were performed at room temperature in darkness. Fluorescence Minus One (FMO) controls of the four analyzed cell markers (CD27, CD38, HLA-DR, and Ki-67) were included in each run to accurately distinguish negative from positive populations. Samples were resuspended in 100 μl of PBS-0.1%BSA (Bovine Serum Albumin, Sigma Aldrich) and acquired in a BD LSRFortessa flow cytometer (BD Bioscience) using FACSDiva software (BD Biosciences) with compensated parameters.

CellROX Deep Red Flow Cytometry Assay Kit (Invitrogen Molecular Probes) was used to evaluate ROS production. Briefly, 5 × 10⁵ harvested cells were stained with CellROX Deep Red Reagent (1 µM) for 25 min at 37°C. For surface staining, cells were stained with anti-CD19-PE-Cy7 and anti-CD27-BV605 (all from BD Biosciences) for 15 min at 37°C. Finally, cells were stained with SYTOX Blue Dead Cell Stain (200 nM) for 15 min at 37°C to identify dead (SYTOX⁺) and viable (SYTOX⁻) cells. ROS production was evaluated as the percentage of viable naïve (CD19⁺CD27⁻) or memory (CD19⁺CD27⁺) B cells that stained positive for CellROX Deep Red probe (CellROX⁺ cells) (Figure 2A).
ROS fold increase induced by each single stimulus related to the unstimulated sample was expressed as a ratio: (single stimulus % CellROX⁺ cells)/(unstimulated % CellROX⁺ cells).
In vitro basal cell death was evaluated as the percentage of SYTOX⁺ naïve (CD19⁺CD27⁻) or memory (CD19⁺CD27⁺) B cells in unstimulated 24-h cultures (Supplementary Figure 1A).

Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).