Investigation of the usability of the 2 ', 7'- dichlorofluorescein diacetate (DCFHDA) probe used for the measurement of tissue free radical level in A tissue-free in vitro environment

Free radicals are very difficult to detect in biological and non-biological environments due to their unstable, short-lived and reactive nature. Fluorescent probes are one of the most preferred methods for the detection of reactive species due to their high sensitivity, ability to pass through the cell membrane and ease of data acquisition.In a previous study in our department using rat cortical slices, 2', 7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent compound was used to measure the level of reactive oxygen species (ROT) in the incubation medium, but unexpectedly, this probe also showed fluorescence in the tissue-free in vitro physiological environment. A significant increase in density was observed. Based on this result, the possible mechanism of the increase in fluorescence intensity and its dependence on ambient conditions (concentration, pH, temperature, time, etc.) were tested. Then, the in vitro usability of the method was tested by looking at the effect ofsome antioxidant compounds on the increase in fluorescence intensity.After re-observing the increase in fluorescence intensity depending on the hydrogen peroxide concentration; It was tested how the ambient pH, oxygen, temperature, incubation time and the fluorescence intensity of the substances in the incubation medium affected. While it was observed that the pH of the environment shifted to basicity, the incubation time and the presence of oxygen increased the fluorescence intensity, it was determined that sodium bicarbonate in the physiological Krebs composition was responsible for the observed effect. On the other hand, the effect of some antioxidant compounds added to the incubation medium on the fluorescence intensity was investigated, and the applicability of the method in tissue free in vitro conditions was tested.In conclusion, the findings of our study a) DCFH-DA, which is mainly used for the detection of reactive oxygen species in the intracellular environment, can also be used in a tissue-free environment, b) but it is closely affected by the incubation conditions, c) therefore, the experimental conditions indicates the importance of standardization.