Intracellular Cytokine Profiling of Human B Cells

Intracellular cytokine staining (ICS) of human B cells was performed as previously described^{41 (link)}. Briefly, cells were stained with LIVE/DEAD fixable Aqua dead cell stain (Thermo Fisher Scientific) for 20 min on ice following which cell-surface marker staining was performed using mouse anti-human CD20 (BD Biosciences; clone: 2H7) and mouse anti-human CD3 (BD Biosciences; clone: UCHT1). Cells were then fixed and permeabilized using fixation/permeabilization buffer (BD Biosciences). Rat anti-human GM-CSF (clone: BVD2-21C11), rat anti-human IL-10 (clone: JES3-19F1) and mouse anti-human TNF (clone: MAb11) antibodies (BD Biosciences) or matching isotype controls were added and cells were incubated for 30 min on ice. Cells were washed and resuspended in FACS buffer (PBS/1%FCS) until analysis on a FACS LSR Fortessa (BD Biosciences).