CD47-mediated neuronal death activates oxidative stress in a caspase-independent pathway. After treatment with 10 μg/mL TSP or 100 μg/mL of 4N1K for different times, ROS was detected with the CM-H2DCFDA using the flow cytometry in primary mouse cortical neurons. (a) Representative fluorescence of ROS. Black line, 0 h group; dotted line, 6 h of 4N1K-treated group; (b) Bar graph of the fluorescence intensity of ROS (mean ± SD); n = 3 independent experiments; *p < 0.05 compared with 0 h group; (c) Pre-treatment with U83836E reduced 4N1K-induced cell death; n = 3 independent experiments performed in triplicate; **p < 0.01 compared with 4N1K-treated group. (d) After treatment with 100 μg/mL 4N1K for 24 h, cleavage of caspase 3 increased (lane 2). Pre-treatment with pan caspase inhibitor (z-VAD-fmk, 40 μM) inhibited the caspase 3 activation triggered by 4N1K (lane 3). However, ROS scavenger U83836E (5 μM) can not inhibit caspase 3 activation (lane 4); (e) Combination pre-treatments with z-VAD-fmk (40 μM) plus U83836E (UE, 5 μM) did not yield additive neuroprotection. n = 3 independent experiments performed in triplicate; *p < 0.05 and **p < 0.01 compared with 4N1K-treated group.