OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 5 × 105 per well were seeded.	- Cells were treated at 37 °C for 60 min with DCF dye. - Cells were treated and incubated with 12AC2O, 3O, 4O, 5O or 6O, each at 10 µM, in a 12-well plate at 37 °C for 8 h

Upstream tips
- 5 × 105 per well were seeded.
Protocol tips
- Cells were treated at 37 °C for 60 min with DCF dye. - Cells were treated and incubated with 12AC2O, 3O, 4O, 5O or 6O, each at 10 µM, in a 12-well plate at 37 °C for 8 h

Publication protocol

In order to detect intracellular ROS in K562 cells treated with 12AC3O, we used an OxiSelect Intracellular ROS Assay Kit (Cell Biolabs, San Diego, CA, USA). First, K562 cells (5 × 105 per well) of K562 were treated at 37 °C for 60 min with DCF dye in the cell culture medium. Then, the cells were washed twice with PBS. After that, the K562 cells were incubated with 12AC2O, 3O, 4O, 5O or 6O, each at 10 µM, in a 12-well plate at 37 °C for 8 h. After removal of the media by centrifugation and washing of the cells with PBS, the cells were next re-suspended and incubated in RPMI medium with cell lysis buffer for 5 min. Fluorescence intensities were quantified by use of a Varioskan Flash (Thermo Scientific, Waltham, MA, USA) at 480/530 nm with a 530-nm cutoff. All samples were prepared in triplicate, and data were presented as the mean ± SD from 3 experiments. Unpaired two-tail t tests were performed to determine significance of differences.

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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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