HCT116 cells were seeded in 96-well black microplates (30,000 cells/well in 200 μL of medium) and were allowed to adhere for 24 h before assay. Then, cells were incubated for 45 min at 37 °C with 1 µM of Dihydrorhodamine 123 (DHR123, Thermo Fisher Scientific, Waltham, MA, USA) prepared in PBS. Then, they were rinsed, and cells were exposed to digested and non-digested Ag NPs and HEC diluted in DMEM F12K medium (containing 1% FBS). Luperox tert-butyl hydroperoxide (Merck Sigma-Aldrich, Saint Quentin Fallavier, France, #458139) at a concentration of 250 µM served as a positive control. The onset of rhodamine fluorescence at λexc/λem 505/540 nm, reflecting cleavage of DHR123 by intracellular reactive oxygen species (ROS), was then monitored at 30 min, 1, 3, 5, 7, and 24 h post-exposure using an ID3 spectrofluorometer (Molecular Devices, San Jose, CA, USA).
Dhr123
Manufactured by Molecular Devices
Sourced in United States
The DHR123 is a high-performance spectrophotometer designed for accurate and reliable absorbance measurements in life science applications. It features a wide wavelength range, a high-resolution detector, and advanced optics for sensitive and precise readings.
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2 protocols using dhr123
1
Intracellular ROS Detection by Dihydrorhodamine 123
2
Measuring Intracellular and Mitochondrial ROS
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Intracellular and mitochondrial reactive oxygen species (ROS) were respectively examined using dihydroethidium (DHE, Cayman, USA)17 (link) and dihydrorhodamine 123 (DHR123, Cayman, USA).18 (link) Cell were loaded with 5 µM DHE and 10 µM DHR123 for 30 min and then treated with amyloid beta alone or in combination with NOX inhibitors and/or MTAs. After treatments, ROS generation was monitored in a SpectraMax Gemini XPS microplate reader (Molecular devices, USA) with excitation at 530 nm and emission at 590 nm for DHE or excitation at 500 nm and emission at 536 nm for DHR123. Each fluorescence value was obtained by subtracting the mean background value of the sham-treated control cultures. In some cases, cultures were viewed under fluorescence microscopy and photographed.
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