Enzymatic Hypoxia Induction Protocol

Hypoxia was induced by enzymatic oxygen depletion using 4 U/ml glucose oxidase and 120 U/ml catalase as previously described [18 (link)]. For this study, the concentration of hypoxia-inducing enzymes was optimised after measuring oxygen levels in cell culture medium using the OxyMini fibre optic oxygen meter (World Precision Instruments, USA, Sarasota) and OxyMicro Software v.00 04/2003. After hypoxia induction, cell culture plates were put in an airtight chamber (Billups-Rothenberg, Del Mar, CA, USA), flushed with nitrogen until the oxygen concentration was < 1% and kept in the incubator at 37 °C for 24 h.