Flow cytometry

Mouse spleens were forced through a 70-µm mesh into FACS buffer (PBS containing 2% heat-inactivated FBS and 2 mM EDTA), and red blood cells were lysed in ammonium-chloride-potassium buffer lysing buffer (Gibco) for 3 min. Cultured cells were harvested by centrifugation. Then cells were washed and Fc-receptors blocked for 15 min on ice. Cells were stained for 20 min on ice with antibodies or reagents listed in Table S4 and, depending on the stain, washed again and secondary-stained for another 20 min on ice before acquisition on a BD LSRFortessa. Anti-idiotype 3BNC60^SI (iv8) produced as human IgG1/κ was detected with anti-human Igκ-BV421 on edited mouse B cells. GC B cells were gated as single/live, B220⁺, CD38⁻ FAS⁺, GL7⁺, and IgD⁻. Allotypic markers CD45.1 and CD45.2 were used to track adoptively transferred B cells.