Flow cytometry and serum antibody analysis of in vivo responses of NP-specific B cells to sialylated and unsialylated conjugates. Rag1−/− mice that received 107 isolated splenic QM transgenic B cells were challenged with 40 µg of the indicated conjugates 2 h later. From the time of reconstitution they were labeled with BrdU as indicated in Materials and methods. Similar results were obtained in two independent experiments similar in design but using CFSE-prelabeled B cells rather than BrdU to measure cell division (e.g., Fig. S4). (A) At day 7 after reconstitution, spleen cells were analyzed for BrdU uptake and B cell marker expression. A lymphocyte gate was used for analysis. Plots shown were representative of mice receiving NP–PA–bNeuGc (n = 5), NP–PA (n = 5), PA–bNeuGc (n = 3), and PBS (n = 3). Percentages are shown. (B) Quantitation of percentages of IgM+ plasma cells as defined by high levels of cytoplasmic IgM. (C) Percentages of B220+ cells scoring positive for BrdU uptake. (D) Serum anti-NP IgM and IgG3 antibody titers of the indicated recipients obtained at day 7 after reconstitution/challenge. Shown are means + SD. *, P < 0.05; and ***, P < 0.005 using the two-tailed Student’s t test. n.s., not significant.
