Fig. 3. RtxA but not proRtxA is cytotoxic against various cells.
a Different cell types (1 × 106/ml) were incubated with increasing concentrations (0.5, 1, 2, and 4 µg/ml) of purified RtxA or proRtxA in the presence of 2 mM calcium ions for 1 h at 37 °C. b HLaC-78 cells (1 × 106/ml) were incubated with the indicated concentrations of purified RtxA for different times at 37 °C. a, b Cell viability was determined by a cell viability staining assay using 1 µg/ml of Hoechst 33258 followed by flow cytometry. The viability of cells incubated without RtxA was reported as 100%. Each point represents the mean value ± SD of four independent experiments. c Erythrocytes (5 × 108/ml) were incubated at 37 °C in the presence of increasing concentrations (0.1, 0.2, and 1 µg/ml) of purified RtxA or 1 µg/ml of proRtxA. Erythrocytes incubated with 1 µg/ml of RtxA in the absence of calcium ions were used as negative control. Cytolytic (hemolytic) activity was measured at different times as the amount of released hemoglobin by photometric determination (A541nm). Complete lysis of erythrocytes was reported as 100%. Each point represents the mean value ± SD of three independent determinations performed in duplicate