Microplate spectrofluorometer

The change in fluorescence resulting from the oxidation of the fluorescent probe H₂DCFDA was used to evaluate the level of intracellular ROS (19 (link)). After RSV and KRGE treatments, the cells were incubated with 50 mM of the fluorescent probe H₂DCFDA. The degree of fluorescence was detected at an excitation and emission of 485 and 535 nm, respectively, using a microplate spectrofluorometer (Molecular Devices Corp., Sunnyvale, CA, USA). Coverslip-loaded 6-well plates were used for image collection during cell culture. After various treatments, H₂DCFDA solution was added to each well of the plate, which was incubated for 2 h at 37°C. Images of the stained cells were collected using a fluorescence microscope (Nikon, Melville, NY, USA).

ONOO⁻ scavenging activity was assessed using a modified Kooy’s method that involves the monitoring of highly fluorescent rhodamine 123, which is rapidly produced from non-fluorescent dihydrorhodamine (DHR) 123 in the presence of ONOO⁻ [37 (link)]. In brief, the rhodamine buffer (pH 7.4) consisted of 50 mM sodium phosphate dibasic, 50 mM sodium phosphate monobasic, 90 mM sodium chloride, 5.0 mM potassium chloride, and 100 μM diethylenetriamine pentaacetate. The final DHR 123 concentration was 5 μM. The assay buffer was prepared prior to use and placed on ice. Test samples were dissolved in 10% DMSO (100 μM final concentration). The background and final fluorescent intensities were measured 5 min after treatment with and without the addition of authentic ONOO⁻ (10 μM), dissolved in 0.3 N sodium hydroxide. The fluorescence intensity of oxidized DHR 123 was evaluated using a microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) at excitation and emission wavelengths of 480 and 530 nm, respectively. Values of ONOO⁻ scavenging activity were calculated as the final fluorescence intensity minus the background fluorescence via the detection of DHR 123 oxidation. L-penicillamine was used as a positive control.