Cd27 pe cf594 m t271

PBMCs were rested after thawing for 4 h in RPMI 1640 medium (Thermo Fisher Scientific) without supplements and subsequently incubated in RPMI 1640 containing live/dead near-infrared dye (Life Technologies) for 20 min at room temperature. Cells were washed, split into two wells, and resuspended in either only RPMI 1640 for the unstimulated sample or RPMI 1640 containing 10 µg/ml goat anti-human IgG/IgA/IgM f(ab)2 (Jackson ImmunoResearch). Stimulation was done for 5 min including a 2-min centrifugation step to remove the medium, followed by addition of the fixation and permeabilization solution of the Foxp3 staining kit according to the manufacturer’s instructions (eBiosciences). Cells were fixed and permeabilized for 45 min at 4°C and then stained with antibodies including CD3 APC-Cy7 (SK7; BioLegend), CD10 PE-Cy5 (HI10a; BD Biosciences), CD14 APC-Cy7 (HCD14; BioLegend), CD16 APC-Cy7 (3G8; BioLegend), CD19 BV510 (SJ25C1; BD Biosciences), CD21 BV711 (B-ly4; BD Biosciences), CD27 PE-CF594 (M-T271; BD Biosciences), IgD APC (IA6-2; BioLegend), and total phosphor-tyrosine PE (PY20; BD Biosciences) for 30 min at room temperature. Cells were extensively washed and measured using a BD Fortessa. Analysis was done with FlowJo 10 (TreeStar).

To determine the frequency of apoptotic cells, we analyzed two major events during apoptosis using flow cytometry: cleavage of caspase 3 and the exposure of phosphatidylserine on the outer leaflet of the cell membrane (Segawa et al., 2014 (link)). Cryopreserved PBMCs from 10 healthy controls and 10 chronically infected patients were analyzed for the frequency of apoptotic cells within the different B cell subsets (Table S2). PBMCs were stained for 30 min with antibodies against surface markers including CD19 biotin (HIB19; BD Biosciences), CD21 PE (B-ly4; BD Biosciences), CD27 PE-CF594 (M-T271; BD Biosciences), IgD PE-Cy7 (IA6-2; BioLegend), and the apoptosis markers cleaved caspase 3 inhibitor FITC-C6-DEVD-FMK (AAT Bioquest). Subsequently, live/dead near-infrared dye (Life Technologies) and streptavidin BV421 (BioLegend) staining was performed for 30 min followed by staining with Annexin V APC (BD Biosciences) according to the manufacturer’s protocol. Samples were analyzed on either a FACS Aria III or a BD FACS Verse immediately after staining and analyzed with FlowJo version 10. Apoptotic cells were defined as double positive for Annexin V and FITC-C6-DEVD-FMK (Jayaraman, 2003 (link)) after dead cell exclusion using live/dead near-infrared dye.