Image-IT™ LIVE Green Reactive Oxygen Species Detection Kit, for microscopy

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- cells were grown on glass cover slips placed in 12-well plate	- Cells were treated with 12.5 µM and 25 µM concentration of HHQ for 48 h

Upstream tips
- cells were grown on glass cover slips placed in 12-well plate
Protocol tips
- Cells were treated with 12.5 µM and 25 µM concentration of HHQ for 48 h

Publication protocol

The reactive oxygen species were detected by fluorescent staining using the Image-iTTM LIVE Green Reactive Oxygen Species (ROS) Detection Kit (Molecular Probes Inc, USA). The assay is based on a nonfluorescent and cell permeable 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). The carboxy-H2DCFDA permeates the live cells and is deacetylated by intracellular esterases. The reduced fluorescein compound is oxidized by the cellular ROS and emits bright green fluorescence with excitation/ emission maxima of 495/529 nm. The cells were grown on glass cover slips placed in 12-well plate and were treated with 12.5 µM and 25 µM concentration of HHQ for 48 h. Subsequently, cells were fixed and then stained for ROS by following manufacturer's instructions. The images were analyzed with AxioVision software (version 4.4, Carl Zeiss).

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