Real-time detection of hypoxia using Image-iT® Hypoxia Reagent

A549, HeLa, or O2OS cells were grown on Nunc™ 30 mm glass bottom dishes in complete medium. The cells were incubated in Flurobrite™ DMEM with 5 µM Image-iT® Hypoxia Probe at 2.5% oxygen (hypoxic) for 1 hr on an EVOS® on-stage incubator and incubated for another 1 hr after restoring oxygen levels to 20% (normoxic). The images were taken on an EVOS® FL Auto Imaging System.