Anti cd19 percp

Staining and flow cytometric analysis was performed as described before [10 (link)]. The Following monoclonal antibodies were used in this study: Via probe PerCP, anti-CD19 PerCP, anti-CD14 PerCP and anti-CD3 PerCP (BD Biosciences, CA, USA) to exclude dead cells, B cells, monocytes and T cells, respectively, and anti-CD56 PC7 (Beckman Coulter, CA, USA) and anti-CD16 APC-H7 (BD Biosciences, CA, USA) to identify NK cells. Additional antibodies that were used include: anti-CXCR3 APC (BD Biosciences, CA, USA), anti-CD69 PE (Invitrogen, CA, USA), anti-CD127 PacBlue (BD Biosciences, CA, USA), anti-CD95 APC (Biolegend, CA, USA), anti-NKp30 APC (Beckman Coulter, CA, USA), anti-NKp46 PE (Beckman Coulter, CA, USA), anti-NKG2D APC (BD Biosciences, CA, USA). At least 1 million events were acquired for each sample, using the BD Canto II (BD Biosciences, CA, USA). Data were analysed with FlowJo 8.8.4 (TreeStar, Or, USA). Lymphocytes were defined by forward and side scatter. CD3⁺, CD14⁺, CD19⁺, dead cells and cell aggregates were removed from analysis based on PerCP and Viaprobe cell viability staining and pulse width analysis. Fluorescence minus one (FMO) staining was used to determine threshold values for the expression of each surface marker.

PE-conjugated DRB1*0301/YFV ENV43-59, DRB1*1101/YFV373-389, DRB1*1501/YFV ENV457-473 tetramers were used for examining YFV ENV-specific cells. PE-conjugated DR1101/FLU HA161-189 and DR1501/FLU HA433-452 were used for examining FLU HA specific CD4⁺ T cells. Staining protocol is similar to those of metal tagged tetramers. Cells were pretreated with dasatinib and were then stained with PE-tetramers at RT for 120 min. Cells were then incubated with anti-PE beads at 4 °C for 20 min. PE-tetramer positive cells were enriched through a magnetic column. Cells were than stained with anti-CD4-APC-H7, anti-CD45RA-v500, anti-CXCR5-APC, anti-CD14-PerCP, anti-CD19-PerCP and Via-probe (all BD Biosciences) for 20 min at 4 °C, washed and then analyzed on a BD LSRII.

After 4 h of stimulation, PBMCs were stained with a combination of the following antibodies from BD Bioscience: anti-CD19-PerCP (cat #345–778), anti-CD43-FITC (cat #555–475), anti-CD27-PECy7 (cat #560–609), anti-CD24-FITC (cat #555–427), anti-CD38-PECy7 (cat #335–825), anti-CD25 FITC (cat #555–431), anti-IL-10 APC (cat #554–707) or anti-IL-10-PE (cat #559–330). Anti-CD5 APC was from Dako (cat #C7242, Glostrup, Denmark) and anti-TIM-1 PE was purchased from Biolegend (cat #353–904).
After 48 h of stimulation, IL-10 was detected with the following BD Bioscience Abs: anti-CD19 APC (cat #555–415), anti-CD14 FITC (cat #555–397), anti-CD4 PerCP (cat #345–770), anti-CD8 PECy7 (cat #557–746) and anti-IL-10 PE (cat #559–330). Live/Dead Fixable Near InfraRed staining (cat #L10119; Molecular Probes, Invitrogen, Carlsbad, CA) was included.
The cells were acquired with a FACS Canto (BD Bioscience) flow cytometer with argon laser (488 nm) and Helium-Neon laser (633nm) excitation.
All analyses were carried out using FlowJo V10 (TreeStar, Ashland, OR). Dead cells were excluded based on Live/Dead Fixable Near InfraRed staining and B cells were identified as CD19⁺ events within a morphological lymphocyte gate. Individual IL-10⁺ B cells were identified using the gating strategy demonstrated in S1 Fig.