Fig. 5. Storage-enhanced RBC necroptosis depends on ROS and NADPH oxidases. Fresh human RBCs or those stored for 1 week in AS-1 were treated with A.) VLY, B.) ILY, C.) PLY, and D.) LLO. Increased RBC necroptosis seen following storage is prevented by inhibition of ROS with N-acetylcysteine (NAC) or NADPH oxidases with VAS2870 but not by inhibition of AGEs with pyridoxamine (PM). Death of fresh RBCs by all toxins tested was prevented by inhibition of ROS with NAC. Hemolysis values were normalized to basal RBC death in the absence of toxin treatment. Results shown are from 3 independent experiments. Two-way ANOVA, ***p<0.001.