Evaluation of the Possibility of Using the Registration of Fluorescence of Stained Cells Isolated from the Skin to Study the Severity of Oxidative Stress

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Abstract

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 in works on 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 intradermical 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 obtained indicate the limitations of the method of staining skin cells with dichlorofluorescein derivatives to assess the intensity of oxidative stress in native tissue.

About the authors

L. A Romodin

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia; National Research Nuclear University MEPhI (Moscow Engineering Physics Institute)

Email: rla2904@mail.ru
Moscow, Russia

A. A Moskovskij

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia; National Research Nuclear University MEPhI (Moscow Engineering Physics Institute)

Moscow, Russia; Moscow, Russia

E. S Chelarskaya

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia

Moscow, Russia

C. C Sodboev

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia

Moscow, Russia

O. V Nikitenko

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia; SSC RF Institute of Biomedical Problems, Russian Academy of Sciences

Moscow, Russia; Moscow, Russia

T. M Bychkova

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia; SSC RF Institute of Biomedical Problems, Russian Academy of Sciences

Moscow, Russia; Moscow, Russia

A. V Mitrofanova

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia

Moscow, Russia

V. I Pustovoit

State Scientific Center of the Russian Federation – Federal Medical Biophysical Center named after A.I. Burnazyan, FMBA of Russia

Moscow, Russia

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