Ficoll paque plus medium

Peripheral blood mononuclear cells (PBMCs) isolation was performed in Ficoll-Paque PLUS medium (VWR, Radnor, PE, USA) using density gradient centrifugation. To get activated B cells, 2 × 10⁶ PBMCs/ml were cultured in DMEM supplemented with 10% FBS, penicillin/streptomycin, and 5 µg PWM/ml at 37 °C for 96 h in 5% CO₂ incubator. Unstimulated PBMCs were grown in the same cultivation medium without PWM under similar culture conditions. Next, cells were divided into aliquots – 2 × 10⁵ cells per aliquot. Each aliquot was stained with anti-CD19 mAb PE-Alexa Fluor 610, anti-CD38 mAb PE/Dazzle 594, and 1 µg of NEF binder or 5 µl of PE anti-CD126 (IL-6Rα) mAb, incubated overnight at 4 °C. For NEF binder’s detection, Streptavidin-PE conjugate antibody was incubated for 30 min at RT. Samples were measured by the Sony SP6800 Spectral Cell Analyzer (Sony Biotechnology, San Jose, CA, USA) and the data was processed using FlowJo V10 software.