CM-H2DCFDA (General Oxidative Stress Indicator)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were pretreated with NAC (10 μM) for 1h.	- Cells after pretreating with NAC are treated with 5 μM CM-H2DCFDA or 10 μM DHE for 30 min at 37 °C. - Tissues were treated with 5 μM CM-H2DCFDA or 10 μM DHE at room temperature for 30 min

Upstream tips
- Cells were pretreated with NAC (10 μM) for 1h.
Protocol tips
- Cells after pretreating with NAC are treated with 5 μM CM-H2DCFDA or 10 μM DHE for 30 min at 37 °C. - Tissues were treated with 5 μM CM-H2DCFDA or 10 μM DHE at room temperature for 30 min

Publication protocol

Intracellular reactive oxygen species (ROS) staining was performed with chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Invitrogen) or dihydroethidium (DHE; Invitrogen) (10–15). CM-H2DCFDA is hydrolyzed by nonspecific esterases to release 2′,7′-dichlorodihydrofluorescein (CM-H2DCF), which is oxidized by intracellular ROS, such as hydrogen peroxide, to CM-DCF, which emits green fluorescence. Upon reaction with superoxide, DHE is converted to ethidium bromide, which binds to DNA and fluoresces red. Cultured cells were loaded with 5 μM CM-H2DCFDA or 10 μM DHE for 30 min at 37 °C. NAC (10 μM) was administered to cells for 1 h before application of CMH2DCFDA and DHE. The freshly frozen tissue sections were incubated in PBS with 5 μM CM-H2DCFDA or 10 μM DHE at room temperature for 30 min. After washing with PBS three times, cells and tissues were observed under an epifluorescence-equipped microscope (Meiji) or a laser confocal microscope (Olympus).

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