OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence)

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	- After adding DCFH solution, plates were incubated for 30 min.

Protocol tips
- After adding DCFH solution, plates were incubated for 30 min.

Publication protocol

A ROS assay kit (OxiSelectTM In vitro ROS/RNS Assay Kit, Cell Biolabs Inc., San Diego, CA, USA) was used to detect ROS. Briefly, the dorsal skin tissue was rapidly frozen using 2-methylbutane and liquid nitrogen. Tissues were homogenized and centrifuged at 10,000 × g for 5 min. Supernatants were removed and stored at −80 °C. In order to quantify both free radicals and hydrogen peroxide (H2O2), separate standard curves for free radicals and hydrogen peroxide were prepared according to the ROS assay kit manual. A standard curve for 2′, 7′-dichlorodihydrofluorescein (DCF) and one for the detection of H2O2 was prepared from a DCF standard (0, 1, 10, 100, 1000, and 10’000 nM DCF) and a 2 mM H2O2 standard (0, 0.039, 0.078, 0.158, 0.313, 0.625, 1.25, 2.5, 5, 10, and 20 μM H2O2), respectively. The skin sample supernatant and H2O2 standard were added to 96-well plates. The catalyst (50 μL of a 1- × dilution) was added and was incubated for 5 min under shaking. A DCFH stock solution (100 μL) was added to each well and was reacted for 30 min in the dark. Fluorescence was read on a fluorescence plate reader (Multilabel counter, VICTOR3TM, Waltham, MA, USA).

For ROS staining, the dorsal skin was rapidly frozen using 2-methylbutane and liquid nitrogen. Cryosections were prepared at a thickness of 20 μm. Sections were incubated with dihydroethidium (DHE; Sigma-Aldrich) in antibody dilution buffer for 2 h at room temperature in the dark. The sections were subsequently washed with phosphate-buffered saline (PBS) and were incubated with DAPI (Vector Laboratories Inc.) in PBS for 10 min. The slides were mounted using Fluorescence-Mounting Medium (Dako, Glostrup, Denmark). An optical microscope (Axio Vision LSM 510, Carl Zeiss Inc.) was used for visualizing fluorescence intensity.

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