Cell proliferation assay with [3H]thymidine incorporation.

A total of 5 × 10⁶ cells/mL was cultured in 96-well flat-bottom plates in the presence of 5, 1.5, 0.5, or 0.15 μg/mL complex antigen preparations obtained from Leptospira (i.e., WCL, secreted proteins, or SDS-soluble protein preparations) or in the presence of recombinant proteins. Medium, concanavalin A at a final concentration of 1 μg/mL, and LPS from E. coli at a final concentration of 5 μg/mL were used as controls. All cultures were performed in quadruplicate and incubated at 37°C with 5% CO₂ for 6 days. Then, 0.25 μCi of [³H]thymidine was added to each well, and cultures were incubated for 24 h. Cells were harvested onto glass fiber filter paper by using a cell harvester, and the incorporation of [³H]thymidine was measured by liquid scintillation counting. Results were measured in counts per minute of [³H]thymidine incorporated into DNA. Results are expressed as mean ± standard error of the mean (SEM) of the counts per minute in quadruplicate cultures.