OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were seeded for 24h and then treated with 1X DCFH-DA and incubated for 60 min at 37 °C	- Cells were treated with Ramizol™ at 0.1 mg mL−1, A-T (10 μM) and stressors 1 mM H2O2, 500 μM menadione and 100 mM glutamate for 60min

Upstream tips
- Cells were seeded for 24h and then treated with 1X DCFH-DA and incubated for 60 min at 37 °C
Protocol tips
- Cells were treated with Ramizol™ at 0.1 mg mL−1, A-T (10 μM) and stressors 1 mM H2O2, 500 μM menadione and 100 mM glutamate for 60min

Publication protocol

Intracellular accumulation of hydrogen peroxide was determined with DCFH-DA. This non-fluorescent compound accumulates within cells upon deacetylation. DCFH then reacts with the reactive oxygen species to form fluorescent dichlorofluorescein (DCF). PC12 cells were plated in 96-well black and clear bottom plates and grown for 24 h before the addition of 1X DCFH-DA, incubation for 60 min at 37 °C, and then treatment with Ramizol™ at 0.1 mg mL−1, A-T (10 μM) and stressors 1 mM H2O2, 500 μM menadione and 100 mM glutamate, respectively for 60 min. Cells were then washed 3 times with Hank's balanced salt solution (HBSS without phenol red) at room temperature. Cellular fluorescence was measured using an Enspire multimode plate reader using an excitation wavelength of 480 nm and emission wavelength of 530 nm (Software version 4.1.) (Perkin Elmer, USA). Results were expressed as Relative Fluorescence Units (RFU).

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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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