Class Switch Recombination: IgM to IgG1

Class switch recombination (CSR) from IgM to IgG1 was performed as previously described [11 (link)]. Naïve B lymphocytes were purified from spleens of 2-month-old mice using EasySep™ mouse B cell enrichment kit (STEMCELL Technology, Vancouver, Canada; #19854), according to the manufacturers’ instructions. For each CSR assay, 2 × 10⁴ cells/200 μL were used in duplicates. The cells were stimulated with LPS (lipopolysaccharides, 40 μg/mL; Sigma Aldrich, St. Louis, MO, USA; #437627-5MG) and IL-4 (Interleukin 4, 20 ng/mL; PeproTech, Stockholm, Sweden; #214-14) for 96 h. Then, the cells were blocked with Fc receptor antibody (2.4G2) and normal mouse serum (Invitrogen, Carlsbad, CA, USA; #10410). The cells were washed in PermWash™ (BD Biosciences, NJ, USA; #554723). Intracellular staining was done using fluorescently tagged anti-mouse antibodies (IgG1-APC) (BioLegend, San Diego, CA, USA; #406610) and the succeeding wash was performed in PermWash. The cells were resuspended in 300 μL of CellFix (BD Biosciences, NJ, USA; #340181). Viable CD19⁺ B lymphocytes were analyzed for IgG1 expression using FlowJo^® (Ashland, Oregon, USA) version 7.6 for Windows.