Abstract

Phenol red (PR) is a commonly used compound in culture media as a pH indicator. However, it is unknown whether this compound can interfere with the pharmacological induction of ferroptosis. Here, using high-content live-cell imaging death analysis, we determined that the presence of PR in the culture medium preconditioned normal and tumor cells to ferroptosis induced by system xc? inhibition mediated by imidazole ketone erastin (IKE) or GPX4 blockade in response to RSL-3, but had no significant effects against treatment with the endoperoxide FINO2. Mechanistically, we revealed that PR decreases the levels of the antiferroptotic genes Slc7a11, Slc3a2, and Gpx4, while promoting the overexpression de Acls4, a key inducer of ferroptosis. Additionally, through superresolution analysis, we determined that the presence of PR mislocalizes the system xc? from the plasma membrane. Thus, our results show that the presence of PR in the culture medium can be a problematic artifact for the accurate interpretation of cell sensitivity to IKE or RSL-3-mediated ferroptosis induction. © 2024