Flow cytometry
APC
Freshly isolated PBMCs were used for phenotypical analysis using the following antibodies: Live/Dead cell marker Near IR (Life Sciences), anti-CD19 (clone SIJ25C1) BUV 395, anti-CD4 (clone SK3) BUV 737, anti-CD16 (clone 3GB) Pacific Blue, anti-CD14 (clone MφP9) AmCyan, anti-Ki67 (clone B56) AF700 (BD Biosciences), anti-CD20 (clone 2H7) FITC, anti-CD123 (clone 6H6) AmCyan, anti-CD27 (clone O323) BV650, anti-CD38 (clone HIT2) BV785, anti-IgD (clone IA6-2) PE-Cy7, anti-IgG (clone HP6017) PE (BioLegend), anti-CD56 (clone N901) ECD, anti-CD3 (clone UCHt1) PE-Cy5 (Beckman Coulter), anti-CD8 (clone 3B5) Qdot 605 (Invitrogen) and anti-IgA (clone IS11-8E10) APC (Miltenyi Biotech). Cells were incubated with 50 μL of surface staining antibody mix diluted in PBS for 30 minutes at 4°C in the dark. Following incubation, the cells were washed twice in FACS buffer (2% FCS and 2 mM EDTA in PBS) and then fixed and permeabilized using Foxp3/Transcription Factor Staining kit (eBioscience) for 30 minutes at 4°C. Cells were then washed twice in permeabilization buffer (eBioscience) followed by intracellular staining. Antibodies against Ki67 and IgG in permeabilization buffer were added to the cells and incubated in the dark for 30 minutes at 4°C. Cells were finally washed with FACS buffer and data acquired on a BD LSR Fortessa (BD Bioscience). Analysis of acquired data was done with FlowJo software version 10 (FlowJo Inc).