Fluorescenční metody pro detekci buněčného poškození

Fluorescent methods are specific, sensitive and selective. These properties are frequently used in scientific studies to detect various cellular parameters. Using fluorescence probes, we are allowed to detect intracellular concentrations of analytes, membrane potentials in cells and organelles or to visualize cell organelles. The aim of our work was to optimize spectrofluorimetric method for routine measurement of glutathione (GSH) concentration in cells. Other partial aims of the study were to optimize the spectrofluorimetric method for the detection of fragmented DNA and to use these and other fluorometric methods to characterize the toxic effect of CdCl2. We used the fluorescent probe monochlorobiman to detect intracellular GSH concentrations. Firstly, we optimized the evaluation of fluorescence intensity signal over time. Consequently, we compared the glutathione concentrations obtained using the optimized calculation method with both the standardly used method and with the GSH levels measured using the reference o-phthalaldehyde method. We found the similarity of the results obtained using optimized and reference methods when we obtained a very strong correlation of both methods in the analysis of biological samples (r = 0.96). For spectrofluorometric detection of fragmented DNA, we used the Hoechst 33258 probe to detect a significant effect of selected model toxic compounds on DNA fragmentation. The highest increase of Hoechst 33258 fluorescence was detected in cells treated with 50 and 100 ěM cisplatin after 24 h. Subsequently, we detected changes in GSH concentration, DNA fragmentation, mitochondrial membrane potential and ROS production in cells incubated with CdCl2.