Carboxy-H2DCFDA (general oxidative stress indicator)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 2 × 10^5 cells/well were seeded. - Cells were either treated with or without diosgenin.	- Cells were incubated with 5 μM carboxy-H2DCFDA for 15 min at 37°C. - Cells were washed with PBS twice, trypsinized, and resuspended in OptiMem I medium.

Upstream tips
- 2 × 10^5 cells/well were seeded. - Cells were either treated with or without diosgenin.
Protocol tips
- Cells were incubated with 5 μM carboxy-H2DCFDA for 15 min at 37°C. - Cells were washed with PBS twice, trypsinized, and resuspended in OptiMem I medium.

Publication protocol

Intracellular generation of ROS was measured with carboxy-H2DCFDA (Invitrogen), which is a cell-permeable and nonfluorescent dye when loaded onto the cells. This compound is oxidized by ROS to fluorescent carboxydichlorofluorescein (DCF) inside the cells. Briefly, the cells seeded in 6-well plates (2 × 105 cells/well) and treated with or without diosgenin were incubated with 5 μM carboxy-H2DCFDA for 15 min at 37°C. Then the cells were washed with phosphate buffered saline (PBS) twice, trypsinized, and resuspended in OptiMem I medium. The fluorescence resulting from the rate of oxidation of the dye in the cells was measured using a FACS with an excitation wavelength of 480 nm and an emission wavelength of 530 nm. The generation of ROS in HepG2 cells was also verified by fluorescence microscopy (Nicon, Japen). Cells grown to confluence were treated with or without diosgenin in the presence of 5 μM carboxy-H2DCFDA for the indicated time and resuspended in fresh OptiMem I medium after washing. During fluorescence imaging, the illumination level was reduced to minimal level to prevent photosensitization of the fluorescent probe.

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