MarkerGene™ Live Cell Fluorescent Reactive Oxygen Species Detection Kit

Protocol tips

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	- 20µM DCFH-DA was treated on cells and incubated at 37°C for 45 min. - Shikonin added to wells at concentration 5µM to generate ROS for 4 Hours.

Protocol tips
- 20µM DCFH-DA was treated on cells and incubated at 37°C for 45 min. - Shikonin added to wells at concentration 5µM to generate ROS for 4 Hours.

Publication protocol

For the assessment of intracellular ROS formation
in cultured breast cancer cells, a fluorescent probe 2´,
7´-dichlorofluorescin diacetate (DCFH-DA) was used.
DCFH-DA was dissolved in PBS at a concentration of 10
mM. To assay ROS, cells were plated in 96 cell well plate. Assay was administered with DCFH-DA (20µM working substrate solution) in culture medium. After incubation at 37°C for 45 min, substrate solution was removed and cells were washed with PBS. Shikonin added to wells at concentration 5µM to generate ROS for 4 Hours. Untreated cells were used as control. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength 528 nm using a fluorescent micro plate reader (BioTek Synergy Ht, Winooski, Vermont, USA). The ROS levels were expressed as RFU (Relative Fluorescence Unit).

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