Characterization of Plasmablasts in Human PBMC

Human PBMC (~5 x 10⁶ live cells) were stained on ice for 30 min with fluorochrome conjugated rHA probes (180–350 pM) in 100 μL of staining buffer (PBS/2% fetal bovine serum [FBS]) as previously described [8 (link), 30 (link), 54 (link), 78 (link), 79 (link)]. Human PBMC were first treated with Fc receptor blocking solution (BioLegend, Dedham, MA, USA) then stained for 30 min on ice using titrated quantities of fluorescently conjugated monoclonal antibodies (S1 Table). After completion of surface labeling, human PBMC were washed extensively with staining buffer prior to fixation with 1.6% paraformaldehyde in staining buffer for 15 min at RT. Following fixation, cells were pelleted by centrifugation at 400xg for 5 min, resuspended in staining buffer and maintained at 4˚C protected from light until acquisition. Data acquisition was performed using the BD FACSARIA Fusion and analysis performed using FlowJo (FlowJo LLC, Ashland, OR, USA). Compensation values were established prior to acquisition using appropriate single stain controls. PBs were defined as CD3/CD14^neg CD19⁺, CD27⁺, CD38⁺⁺ cells as previously described [67 (link), 80 (link)].