Single B cells were cultured in the presence of NB-21.2D9 feeder cells, kindly provided by Dr. Garnett Kelsoe from Duke University (46 (link)). NB-21.2D9 cells were seeded into 96-well plates at a density of 1000 cells per 100 μL/well in B cell medium (RPMI-1640 supplemented with 10% FBS, 55 μM 2-mercaptoethanol, 100 Units/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 1% MEM nonessential amino acid). Next day (day 0), recombinant mouse IL-4 (4 ng/mL, 100 μL/well; Peprotech) was added to the cultures, and single B cells were then sorted directly into each well using a BD FACSAria cell sorter. On day 2, 100 μL of culture medium were removed from the cultures and 200 μL of fresh B cell medium were added. From days 3 to 8, two-thirds of the culture media (200 μL) were replaced with fresh B cell medium every day. On day 10, the culture supernatants were harvested and total IgG levels were determined by ELISA. Additionally, autoreactivity against nuclear and cytoplasmic self-antigens was determined by QUANTA Lite ANA ELISA (Inova Diagnostics).