OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- 1X DCFH-DA/media solution contains 5% methanol. For cells that are sensitive to methanol, we recommend instead preparing a 0.1X (100 µM) solution of DCFH-DA in cell culture media.

Protocol tips
- 1X DCFH-DA/media solution contains 5% methanol. For cells that are sensitive to methanol, we recommend instead preparing a 0.1X (100 µM) solution of DCFH-DA in cell culture media.

Publication protocol

To quantify intracellular ROS, OxiSelect™ Intracellular ROS Assay Kit (Cell Biolabs) was used following the manufacturer’s instruction. Fluorescence intensity was measured with spectrofluorometer plate reader. Standard curve was included in every independent assay. MitoSOX™ Red mitochondrial superoxide indicator (Life Technologies) was applied to determine mitochondrial superoxide level following manufacturer’s instruction. Mitochondria ROS quantification in 96-well format was performed using protocols described elsewhere22. Briefly, MitoSOX reagent in working solution was added to each well after stimulation and incubated at 37 °C in the dark. Lysis buffer was added after three time wash before measurement. The measured fluorescence value was expressed as a fold change compared with that of untreated control. For live cell imaging, MitoSox reagent working solution was loaded to cover cells and incubated at 37 °C in the dark. Cells were then washed gently before imaging acquisition. Nuclei were counterstained with DAPI. Images were captured and processed with Nikon ECLIPSE Ti Inverted Research Microscope and NIH Image J.

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Manufacturer protocol

Download the product protocol from Cell Biolabs for OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) below.

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