Oxidative stress is a key link in the development of many pathological processes. Fluorescent dyes sensitive to reactive oxygen species, in particular dichlorofluorescein and its derivatives, are widely used for its evaluation. These dyes are actively used when working with cell cultures, but there is very little data on their use for the analysis of cells isolated directly from animal tissues. The present study was devoted to the investigation of the possibility of using 6-carboxy-dichlorofluorescein to register oxidative stress in cells isolated from rat skin flaps. To induce reactive oxygen species, skin fragments were incubated in a medium with hydrogen peroxide, after which cell suspensions were prepared from them. The cells were stained with 6-carboxy-dichlorofluorescein and Hoechst-33342, which allowed the fluorescent signal to be normalized by the number of cells in the sample. In separate experiments, the precursor of 6-carboxy-dichlorofluorescein was injected intermediately to live animals. Evaluation of the fluorescence ratio of 6-carboxy-2',7'-dichlorofluorescein and Hoechst-33342 showed the possibility of registering only pronounced oxidative stress, while it was not possible to distinguish its degree or record low-intensity changes. Additionally, microscopic analysis revealed the presence of cellular aggregates and fragments of the intercellular matrix, which reduced the reliability of the results. The data we obtained indicate the limitations of the method of staining skin cells with dichlorofluorescein derivatives to assess the intensity of oxidative stress in native tissue.
Romodin et al. (Wed,) studied this question.