B Cell Class Switch Recombination Assays

Resting splenic B cells were isolated using CD43-microbeads, stained with 5 μM CFSE and cultured for 72 h in vitro with LPS (50 μg/ml) for CSR to IgG3 and IgG2b, LPS + IL-4 (5 ng/ml) for CSR to IgG1 and IgE and LPS + IFN-γ (100 ng/ml) for CSR to IgG2a. CSR was assayed by flow cytometry as described [4 (link)]. Primary B cells were transduced with retroviruses expressing Parp3^Flag, Flag alone or AID^Flag-HA and a GFP reporter as previously described [53 (link)]. For knockdown experiments, the hairpin sequence for AID (5’-ACCAGTCGCCATTATAATGCAA-3’) was cloned into the LMP retroviral vector (Open Biosystems), transductions were performed as previously described [49 (link)].