Additional loss of Bad or Bid did not further increase the abnormally elevated number of high-affinity AFCs and low-affinity AFCs in Bim-deficient mice. Wt, bim^−/−, bim^−/−bad^−/−, or bim^−/−bid^−/− mice were immunized with NP coupled to KLH. After 14 or 28 days, spleen and bone marrow were harvested, and ELISPOT assays were performed to determine the numbers of NP-specific AFCs. Frequencies of NP-specific IgG1⁺-secreting AFCs in the spleen of wt and bim^−/−bad^−/− mice (A, total cell number) or wt, bim^−/− and bim^−/−bid^−/− mice (C, total cell number) and in the bone marrow (B and D, percentage) were determined. The total numbers of AFCs are represented by the overall height of the column (high- plus low-affinity NP-specific AFCs; ie, binding to NP₂₀) and the high-affinity (antibodies binding to NP₂) AFCs are represented by the black proportion of the column. The ratio of NP₂/NP₂₀ antibody producing cells indicates the affinity maturation, and this is shown on top of each column. Data represent the mean of n = 2-3 bim^−/−bad^−/− mice, n = 2 bim^−/−bid^−/− mice, bim^−/− = 3 mice, and n = 3 wt mice.