Dependence of temperature and salt concentration on the transient accumulation of prefibrillar intermediates. The time course for the spontaneous formation of insulin amyloid fibrils in the presence of 1.0 (A and B) or 0.1 m NaCl (C) at different temperatures was monitored using ThT fluorescence. A, the time course for the formation of insulin amyloid fibrils in the presence of 1.0 m NaCl at 90 (red), 85 (orange), 80 (yellow), and 75 °C (light green), and B, 70 (green), 65 (cyan), 60 (dark blue), and 55 °C (purple). C, the time course in the presence of 0.1 m NaCl at 90 (red), 80 (yellow), and 75 °C (light green), in which no abrupt increase in ThT fluorescence intensity was observed immediately after the temperature increase.