Similar proliferative responsiveness of FO and MZ B cells of normal and KLF3 transgenic mice in vitro. (A) FO and MZ B cells were isolated from the spleens of nontransgenic and KLF3 transgenic littermates using magnetic bead enrichment and cell sorting based on CD21/35, CD23, and CD19 staining. A total of 2 × 10⁴ cells were cultured in 0.2 mL medium to which graded amounts of LPS were added as indicated. ³H-Thymidine (0.5 μCi/well) was added on day 2, and incorporated radioactivity was measured on day 3. Assays were performed in triplicate; mean values and error bars (representing SDs) are shown. (B) DNA G₁/G₂/S profiles at 42 hours after stimulation by anti-IgM, anti-CD40 + IgM, anti-CD40 alone, or LPS. (C) CFSE profiles on days 2 to 4 upon LPS stimulation of lymph node cells depleted of Thy-1.2⁺ cells. (D) Increase in cell numbers on days 2 to 4 on LPS stimulation of lymph node cells depleted of Thy-1.2⁺ cells. Cells were counted by FACS using beads of known concentration as reference. (E) Limiting dilution assay for the determination of the frequency of IgM,κ-secreting cells upon LPS stimulation on rat feeder cells. Graded amounts of Thy-1.2⁺ depleted lymph node cells were seeded onto 4 × 10⁵ rat thymus feeder cells in 96-well plates. Mouse IgM,κ secretion was determined by enzyme-linked immunosorbent assay. Wells were counted positive if a given well gave an OD that differed by more than 3 SDs from the mean of negative wells. (A) One of 2 independent experiments is shown. (B-D) The experiment was performed in parallel with transgenic and nontransgenic littermate cells of 2 founder lines, giving similar results, and was repeated once. (E) The experiment was repeated once, giving similar results.