Flow cytometric evaluation of hypoxic cells in solid experimental tumours using fluorescence immunodetection.

Numerous methods have been proposed for the detection of hypoxic cells using nitroimidazoles labelled with both radioactive and stable isotopes where the isotopic label becomes bound as a result of reductive metabolism of the nitro group. A new probe for hypoxia, 7-(4'-(2-nitroimidazol-l-yl)-butyl)-theophylline, is described where an immunologically recognisable hapten (theophylline) is covalently linked to a 2-nitroimidazole. Bioreduction of the nitroimidazole leads to binding of bioreductive metabolites, and hence the theophylline side-chain, to intracellular molecules. Immunochemical procedures are then used to stain cells containing the bound theophylline using an FITC-conjugated anti-serum. Flow cytometric analysis of stained cells is facilitated by co-staining cellular DNA, which allows discrimination of single cells in the sample and rejection of cell clumps and debris. The alternative use of an immunoperoxidase-conjugated anti-serum has been used to demonstrate the localisation of hypoxic cells in frozen tumour sections.