Time course of lethal toxin-induced O
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).