Investigating IFN-α2b Effects on B Cell Viability and Differentiation

B cells were obtained by magnetic cell sorting for cell culture experiments. Purified B cells were cultured in 96-well plates (2 × 10⁵ cells per well) in complete Roswell Park Memorial Institute-1640 medium. To test the effects of interferon on cell viability and apoptosis, varying concentrations of IFN-α2b (PBL Assay Science) were applied and assessed by an Apoptosis Detection Kit (BioLegend). In B cell differentiation experiments, cells were stimulated with anti-IgM (20 ug/mL, Invitrogen), and IFN-α2b (5,000 U/mL), preincubated with or without IFN α-interferon-α/β receptor-inhibitor (IFNAR-IN)-1 hydrochloride (5 μM, MedChemExpress), an inhibitor of IFN-α2b and IFNAR interaction before IFN-α2b treatment.^{23 (link),24 (link)} Following 3-day culture, B-cell subtypes were assessed by FACS. In the last set of experiments, cells were plated in duplicates in the B cell culture medium, at 2 × 105 cells/200 ul in 96-well plates in the presence of Soluble CD40-ligand (sCD40L; 100 ng/ml, R&D Systems), interleukin-2 (IL-2; 50 ng/ml R&D Systems), interleukin- 21 (IL-21; 50 ng/ml R&D Systems), interleukin-6 (IL-6; 5 ng/ml R&D Systems), tumor necrosis factor-α (TNF-α; 5 ng/ml PeproTech), with or without IFN-α2b (5000 U/ml). After 10 days in vitro, culture supernatant was collected to quantify AQP4-IgG, as previously reported.^{4 (link),25 (link)}