FIG. 4.

Antibody response of E/HEL-Tg B cells in vitro. Equal numbers of HEL-Tg and E/HEL-Tg B cells were activated with 10 μg/ml hen egg lysozyme and 1 μg/ml anti-CD40 antibody for 3, 5, or 7 days at 37°C with 5% CO₂. (a) Supernatants from individual wells for each time point were harvested and analyzed for the production of cumulative HEL-specific IgM by ELISA as described in Materials and Methods. The data are an average of the optical density (OD) readings of multiple wells for HEL-specific IgM ± standard error. (b and c) The total cell number and percent viability were determined by trypan blue exclusion at the indicated time points. (d) HEL-Tg and E/HEL-Tg B cells were activated for either 3 or 5 days, and viable cell numbers were determined. Equal numbers of viable cells were plated for ELISPOT analysis for 4 h as described in Materials and Methods. The percentage of plasma cells was determined by the following equation: (no. of spots counted/no. of cells plated) × 100. The data are an average of multiple wells ± standard error. The percentage of plasma cells was not determined on day 7 by ELISPOT analysis due to the lack of an adequate number of viable cells for the assay. For panels a to d, the data are representative of two to three experiments with similar results.