ROS-ID® Total ROS/Superoxide detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were pre treated with 8.45 μm JAHA	- After 24 and 48 h of incubation and resuspended in 500 μL of ROS/ Superoxide Detection Mix and incubated for 30 min at 37 °C in the dark

Upstream tips
- Cells were pre treated with 8.45 μm JAHA
Protocol tips
- After 24 and 48 h of incubation and resuspended in 500 μL of ROS/ Superoxide Detection Mix and incubated for 30 min at 37 °C in the dark

Publication protocol

The production of ROS, such as hydrogen peroxide, peroxynitrite, hydroxyl radicals, nitric oxide, and peroxy radical, and of superoxide was checked using the Total ROS/ Superoxide Detection Kit (Enzo Life Sciences, Lausen, Switzerland) according to the manufacturer's instructions. Briefly, attached and floating MDA-MB231 cells treated with 8.45 μm JAHA were harvested after 24 and 48 h of incubation and resuspended in 500 μL of ROS/ Superoxide Detection Mix and incubated for 30 min at 37 °C in the dark prior to flow cytometry assay for the green and orange fluorescence in a FACSCanto apparatus. Vehicle treated cells were tested as parallel controls. Positive (pyocyanin-treated) and negative (N-acetyl-L-cysteine-treated) controls were included in the analysis, and data were represented as dot plots using Flowing software v.1.6.0., which discriminate cells with increased total ROS production (top left quadrant), increased superoxide production (bottom right quadrant), and increased total ROS and superoxide production (top right quadrant). To assess the involvement of ROS production in the cytotoxic effect of JAHA, an MTT assay was performed as already reported on JAHA-treated cells incubated for 24 h in the presence of the antioxidant butylated hydroxytoluene (BHT)16 at 5, 10, 25, and 50 μm final concentration.

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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for ROS-ID® Total ROS/Superoxide detection kit below.

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