EF-5 (2-Nitroimidazole) is a agent of hypoxia labeling. It is used to identify hypoxia in cells.

EF-5 binding and metabolic reduction of the prodrugs are highly correlated in a panel of 14 hypoxic tumor cell lines. Overexpression of CYPOR causes similar 2- to 4-fold increases in EF-5 binding and metabolic reduction of tirapazamine and CEN-209 in SiHa and HCT116 cell lines. Which is similar enhancement of γH2AX formation.[1].

EF-5 binding is a promising stratification biomarker for benzotriazine-N-oxide bioreductive prodrugs. Binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent, following intravenous injection of EF-5. 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]. 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. The distribution of hypoxia in a tumor can be inferred by examining individual tumor cells using flow cytometric techniques[2].