EF-5 (EF5; 2-Nitroimidazole) is a hypoxia labeling agent used to identify hypoxia in cells. In Vitro: Overexpression of CYPOR induces similar 2- to 4-fold increases in EF-5 binding and metabolic reduction of tirapazamine and CEN-209 in SiHa and HCT116 cell lines, and similar enhancement of γH2AX formation. EF-5 binding and metabolic reduction of the prodrugs are highly correlated in a panel of 14 hypoxic tumor cell lines^[1]. In Vivo: EF-5 binding is a promising stratification biomarker for benzotriazine-N-oxide bioreductive prodrugs. In HCT116 xenografts, CYPOR overexpression also significantly increases EF-5 binding and CEN-209 reduction, and modification of tumor hypoxia causes similar changes to the bioreductive activation of both agents, resulting in a strong correlation between EF-5 binding and CEN209-induced DNA damage at the individual tumor level^[1]. Following intravenous injection of EF-5, binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent. Detection of binding using fluorescence microscopy can be performed on frozen tissues; tissue sections can be counterstained with haematoxylin and eosin for light microscopic analysis. Alternatively, the distribution of hypoxia in a tumor can be inferred by examining individual tumor cells using flow cytometric techniques^[2].