The effect of PU.1 deletion on B-cell function. (A) Immunoglobulin isotype levels in untreated mice. Sera from 8- to 12-week-old mice (n = 6 for each genotype) were analyzed for levels of IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, Igκ, and Igλ by ELISA. PU.1^+/+CD19 and PU.1^Δ/ΔCD19 are shown in black and white circles, respectively. (B-C) Number of NP-specific IgG1 ASCs in bone marrow and spleen. PU.1^+/+CD19 and PU.1^Δ/ΔCD19 mice were immunized with 100 μg NP-KLH precipitated in alum and boosted at day 28 with 20 μg soluble NP-KLH. On the indicated day, spleens (Sp) and bone marrow (BM) were harvested and a known number of cells were cultured overnight on ELISPOT plates with high (B) or low (C) conjugation ratio NP-specific coat. Bound antibody was detected by incubating with IgG1-HRP and spots revealed with AEC substrate. The number of PU.1^+/+CD19 and PU.1^Δ/ΔCD19 ASCs are shown in gray and white, respectively. Numbers represent the mean ± standard deviation. (D) Germinal center formation. Spleens from mice were collected at day 7 and 28 following immunization. Sections were cut and stained for the germinal center B cells (GL7, red/brown) and follicular B cells (IgD, blue).