ROS-Glo™ H2O2 Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 10,000 MDA-MB-231 cells per well in a total volume of 70 µl.	- After adding the test compunds and H2O2 solution, the plate was incubated at 37°C in a 5% CO2 incubator for 60 min. - After adding ROS-Glo detection solution, plate was incubated for an additional 20 min

Upstream tips
- 10,000 MDA-MB-231 cells per well in a total volume of 70 µl.
Protocol tips
- After adding the test compunds and H2O2 solution, the plate was incubated at 37°C in a 5% CO2 incubator for 60 min. - After adding ROS-Glo detection solution, plate was incubated for an additional 20 min

Publication protocol

We measured H2O2 production with the ROS-Glo™ H2O2 Assay system. All compounds were tested at 10 µM in PBS or MEM plus or minus 1 mM sodium pyruvate. Compounds were tested in triplicate and each experiment was performed a minimum of three separate times. Stock solutions were prepared as follows: 10 mM menadione in DMSO, 70 mM pyrogallol in DMSO, 40 mM benserazide in DMSO, 10 mM eseroline in DMSO, and 10.2 mM alamethicin in ethanol. Vehicle was present at 0.1% in these experiments. The H2O2 assay was performed as directed by the manufacturer as follows. The compounds were added to an opaque white 96 well plate in the desired media or PBS. The H2O2 substrate solution was then added, bringing the final volume to 100 µl. The plate was incubated at 37°C in a 5% CO2 incubator for 60 min. 100 µl of the ROS-Glo detection solution was added to each well at the end of the incubation. After an additional 20 min incubation at room temperature, luminescence was recorded using a GloMax® Multi Detection System luminometer.

To confirm the selectivity of ROS-Glo for H2O2, 35 U of catalase was included in some of the 100 μl reactions. In order to estimate the amount of H2O2 generated by these compounds, a standard curve was also performed at the same time as the corresponding experiment. Concentrations between 0 and 30 μM H2O2 were used to construct the standard curve. A standard curve in MEM media was used to quantitate the data using MEM media or MEM with pyruvate and a standard curve in PBS was used to quantitate the data in PBS. Due to the large difference in background between samples in MEM, samples in MEM with pyruvate, and those containing catalase, all data points and the standard curve were background corrected using the appropriate controls. The equation of the linear line of the curve was then used to determine the amount of H2O2 generated in each sample.

Assays to determine the production of H2O2 in the presence of cells were performed as described above with the following modifications. The day before the experiment, cells were plated in clear, tissue culture treated 96-well plates at a density of 10,000 MDA-MB-231 cells per well in a total volume of 70 µl. Wells with cells were paired with corresponding wells without cells (medium alone). After overnight incubation at 37°C in a 5% CO2 incubator, 10 µl of compounds or their vehicle in the appropriate medium were added, followed by 20 µl of the H2O2 substrate solution for a total volume of 100 µl. After the 1 h incubation at 37°C in a 5% CO2 incubator, 50 µl of each reaction mixture was transferred to a white 96-well plate and mixed with 50 µl of the ROS-Glo detection solution. The plate was then incubated 20 min at room temperature before reading luminescence. The standard curve for these experiments was performed analogously to the experiment. Briefly, the incubation of the H2O2 samples occurred in a cell culture treated plate, after which 50 µl was moved to a white luminometer plate and mixed with 50 µl of detection reagent.

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