[Fluorescent formazans in flow cytometry. Studies of their oxygen sensitivity]

In order to supply the tetrazolium salt procedure to flow cytometry we have synthesized monotetrazolium salts which are converted into fluorescent formazans on reduction. In this preliminary study we investigated the "oxygen sensitivity" of four of our compounds and compared them to the commercially available nitrotetrazole blue (NBT). The amount of formazan produced by Ehrlich ascites tumour cells via the succinic dehydrogenase reaction during oxic and anoxic incubations was determined by elution technique. Under oxic conditions no NBT formazan but much cyano-di-chlorophenyl formazan (==CCPC) was generated. The yields of this formazan differed only slightly between oxic and anoxic incubations. Twice as much of the amount of the other three formazans cyano-ditolyl- (==CTC, Stellmach 1984), cyanodiphenyl- (==CPC), and cyano-dianisyl tetrazolium chloride (==CAC), were produced in nitrogen atmosphere as in oxygen. The amount of all formazans investigated increased both in oxic and in anoxic conditions if cyanide was present. All cyano-aryl formazans have the maximum of absorbance between 430 and 470 nm and fluoresce in the spectral range above 580 nm with CTC formazan being the brightest. The tetrazolium salts, i.e. in unreduced state, did not fluoresce in the visible part of the spectrum. The formazans could be applied to flow cytometry. After staining the nuclear DNA with the fluorochrome DAPI, we obtained detailed two-parametric distributions correlating the amount of formazan per cell with the DNA content. The results are a step to establish a method for automated identification of malignant cells.