LPS responsiveness of wt and CD19^-/- B-cell precursors. Wild-type and CD19^-/- BM culture cells were stimulated with 50 μg/mL LPS for 48 hours. (A) Supernatants of stimulated cells were collected and assayed for IgM by ELISA. IgM concentrations were determined using a reference IgM standard curve and are expressed as nanograms per milliliter. ▦ indicates LPS; □, medium. Results are expressed as mean ± SE of 3 different experiments. (B) Visualization and quantification of antibody-producing plasma cells in the stimulated cultures. Cultured cells that were stimulated with LPS for 48 hours were then transferred to filters for analysis of plasma cells by an ELISPOT assay. Frequencies of IgM-producing plasma cells were calculated and expressed as number of IgM-producing cells per 10⁵ precursor cells. ▦ indicates LPS; □, medium. Results are expressed as mean ± SE of 3 different experiments. Representative filters are shown for each bar. (C) Expression of RP105 in early B-cell precursors. Cultured cells were stained for IgM and RP105 and analyzed by FACS. Plots shown are representative of 3 experiments. Actin blot is shown as an internal control gene expression.