ROS-Glo™ H2O2 Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 0, 500, 1000, 5000, and 1000 wild type and autobioluminescent HEK293 cells were platted	- Cells were incubated at 37°C and 5% CO2 for 18 h before the addition of 20 µl 125 µM H2O2 substrate. - each well was then incubated for an additional 6 h followed by bioluminescent measurements of H2O2 levels.

Upstream tips
- 0, 500, 1000, 5000, and 1000 wild type and autobioluminescent HEK293 cells were platted
Protocol tips
- Cells were incubated at 37°C and 5% CO2 for 18 h before the addition of 20 µl 125 µM H2O2 substrate. - each well was then incubated for an additional 6 h followed by bioluminescent measurements of H2O2 levels.

Publication protocol

Autobioluminescent HCT116 cells stably transfected with the pCMVLux vector were seeded in triplicate into 96 well plates at plating volumes of 0, 100, 250, 500, 750, 1000, 2500, and 5000 cells/well in 80 µl volumes of medium. Identical numbers of untransfected wild type cells were similarly plated to act as positive controls. Due to the low observed values generated by wild type HEK293 cells, only platings of 0, 500, 1000, 5000, and 1000 wild type and autobioluminescent HEK293 cells were performed. H2O2 levels were measured using the ROS-Glo assay (Promega) according to the manufacturer's instructions. Each plate of the autobioluminescent pCMVLux-expressing or wild type positive control HCT116 and HEK293 cells was incubated at 37°C and 5% CO2 for 18 h before the addition of 20 µl 125 µM H2O2 substrate (provided in the kit), and each well was then incubated for an additional 6 h followed by bioluminescent measurements of H2O2 levels. Bioluminescent readings were obtained using a SynergyII plate reader (BioTek) with a 1 sec integration time and reported as relative light units. This process was repeated three times to obtain three biological replicates, each consisting of three technical replicates. Relative light units from each plating density were averaged and background values representative of the relative light units detected from the 0 cells/well treatment conditions were subtracted to remove compounding effects from potentially reactive oxygen species in the medium. The average values for each of the triplicate plates were used to determine standard errors of the mean for each assay.

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Manufacturer protocol

Download the product protocol from Promega for ROS-Glo™ H2O2 Assay below.

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