Vacuolating cytotoxic activity of mutant proteins. Wild-type H. pylori strain 60190 and strains expressing mutant VacA proteins were grown in broth culture, and secreted VacA proteins were normalized as described in Methods. Serial two-fold dilutions of VacA-containing preparations were added to HeLa cells (A), RK13 cells (B), and AZ-521 cells (C). Vacuolating activity was measured by neutral red uptake. Relative VacA concentrations are indicated. Results represent the mean ± SD from triplicate samples, expressed as a percent of neutral red uptake induced by wild-type VacA. *, p ≤ 0.02 as determined by Student's t-test compared to wild type VacA. Similar results were observed in three independent experiments.