Priming with GFP does not increase HEL-specific B-cell numbers. (A) Gating strategy for detection of total B cells via flow cytometry. (B) Gating strategy for detection of HEL-specific B cells using dual tetramer staining. Example dot plots show results after incubation of HEL-specific tetramers with a mixture of WT splenocytes and splenocytes from HEL-specific B-cell transgenic MD4 mice or WT splenocytes alone. (C) Quantification of percent HEL tetramer detection of HEL-specific MD4 B cells after mixing with WT splenocytes at defined ratios. (D) Quantification of percent HEL tetramer–positive B cells examined in PBS-treated, GFP-primed or B6-exposed recipients. Error bars represent mean ± SD. FSC, forward scatter; ns, no significance; SSC, side scatter.