Protocol to model tumor hypoxia in vitro using real-time phosphorescence-based sensing of O₂ gradients generated by metastatic cancer cells

Tumor hypoxia plays a critical role in cancer progression and therapeutic resistance. Here, we present a protocol for the self-generation of hypoxia by metastatic cancer cells using phosphorescence-based O₂ sensing. We describe steps for phosphorescent film calibration, system assembly, and real-time imaging of hypoxia development. We then detail approaches to spatially map resulting O₂ gradients. This protocol supports applications for studying key behaviors linked to metastatic progression, including motility and aerotaxis, under physiologically relevant hypoxic conditions. For complete details on the use and execution of this protocol, please refer to Hosny et al.