Monitoring the different changing behaviors of •OH and cysteine in two ferroptosis pathways by a dual-functional fluorescence probe

Ferroptosis is a unique iron-dependent modality of regulated cell death, which attracted growing research interests in the past few years because of its potential pathological roles and therapeutic functions in many critical diseases. Hydroxyl radical (•OH) and cysteine (Cys) play not only crucial but also highly related roles in the development and redox regulation in ferroptosis. Herein, by virtue of a special sequential response strategy, we developed a new dual-functional fluorescence probe Coum-HCy for •OH and Cys. Coum-HCy is capable of selective, sensitive and especially sequential detection of •OH and Cys in two separated fluorescence channels (λem = 650 nm for •OH and λem = 453 nm for Cys). With Coum-HCy, we have monitored the different changing behaviors of •OH and Cys in two ferroptosis pathways initiated by erastin or RSL3 respectively. The results show that •OH is significantly generated during whether erastin or RSL3 initiating ferroptosis. However, due to the distinct mechanism of erastin and RSL3, the intracellular Cys level obviously decrease when cells treated with erastin but keep unaffected by RSL3. The good analytical properties of Coum-HCy may enable it to be widely used in the studies of more ferroptosis-relative physiological or pathological processes, which would not only simplify the detection procedures but also provide more accurate information to indicate the possible mutual correlation between •OH and Cys during ferroptosis.