Fig. 1

Time course of lethal toxin-induced O ₂ ⁻ production and lysis

Mϕ production of O₂⁻ (a) and cytotoxicity (b) were compared after exposure to LeTx or controls. RAW 264.7 cells were grown and prepared as described. Superoxide anion production was determined by monitoring changes in Absorbance₅₅₀ due to cytochrome c reduction. Cells to be monitored for cytotoxicity were loaded with Na₂⁵¹ CrO₄ for 16 hr, and washed twice before challenge. ⁵¹Cr release was normalized for spontaneous leakage (<1,000 cpm/10⁶ cells) and plotted as the percentage of total radioactivity in the cells (approximately 12,000 cpm/10⁶ cells). Challenge concentrations were: PA = 0.1 µg/ml, LF = 0.1 µg/ml, PMA =10 ng/ml. Experiments measuring O₂⁻ production and cytotoxicity were run in parallel (triplicate samples); with three experimental series run for both ⁵¹Cr and O₂⁻ production. SEM values were <13% of the indicated value (O₂⁻ measurement) and <5% of the indicated value (⁵¹Cr assay) (n = 3). Higher doses of PMA (highest dose = 0.1 µg/ml) did not significantly increase O₂⁻ production and did not induce cytotoxicity (data not shown).