Fig. 4. The effect of superoxide scavangers on aggregation and XTT reduction. (A) Ax2 cells were allowed to develop after plating onto plastic dishes at a density of 9.3x10⁵ cells/cm² in buffer alone, or buffer plus 500 units/ml SOD, 1.5 mM MTT, 0.5 mM NBT, 10 mM tiron, or 1 mM XTT. The cells were photographed after 12 hours. The bar represents 0.5 mm. (B) Ax2 cells were developed in tissue culture dishes at a density of 1.4x10⁶ cells/cm² in the presence of the stated concentration of MTT, NBT or tiron, along with 0.5 mM XTT, for 24 hours, when the buffer was collected and the absorbance at 470 nm measured. The data are expressed as percentage reductions in absorbance relative to a control from cells developed in the presence of 0.5 mM XTT alone, and are the means ± s.e.m. of three experiments. Asterisks indicate concentrations of scavengers that were sufficient to inhibit aggregation at this cell density. (C) Exponentially growing Ax2 cells were diluted 1:12 in phosphate buffer and superoxide scavenger added (XTT 1 mM, MTT 1.5 mM, NBT 0.2 mM, tiron 20 mM). These concentrations of scavenger all inhibited aggregation. The cultures were incubated for 18 hours at 22°C, 180 rpm before serial dilution in phosphate buffer and 300 cells of each were plated over 2x 15-cm SM plates, in association with Klebsiella aerogenes. Cell survival was determined by counting the number of clonal Dictyostelium colonies appearing on the bacterial lawn. Data are the means ± s.e.m. of triplicate plates within one experiment. One experiment representative of 3 independent experiments is shown. (D) Exponentially growing Ax2 cells were harvested and plated to aggregate under buffer as described in legend to Fig. 4A, but at three different cell densities, as indicated below. 1.5 mM MTT was sufficient to inhibit aggregation at the two lower cell densities, but not at the highest cell density. Similar density dependence was observed for the inhibition of aggregation in the presence of XTT, NBT and tiron (data not shown).