In vitro analysis of conjugate binding and response by NP-specific B cells. (A) Flow cytometry analysis of conjugate binding to isolated NP-specific B cells from QM transgenic mice. PA ligands used in this assay carried biotin tags along with the indicated carbohydrate and hapten moieties. (B) Ca²⁺ mobilization response monitored by Fluo-4 dye fluorescence. (C) Analysis of proliferation induced by the indicated concentrations of antigens by ³[H]thymidine uptake. Cells were harvested at 39 h of culture after a 15-h labeling period. Shown are means + SD. *, P < 0.05; and ***, P < 0.005 using the two-tailed Student’s t test. (D–G) Western blot analysis of protein phosphorylation induced by the indicated conjugates. The arrow indicates the expected molecular weight of CD22 on the pTyr blot. The number of independent replicates of the experiments shown in the indicated sections were as follows: A, one; B, two; C, three; D, three; and E–G, one.