Other than DAB staining, what is an effective way to detect ROS production. Perhaps a more quantitative way?
Hi Lauren, there is a fluorescent dye called DCF, which can be used for ROS detection. You can use it for quantitative determination of ROS production, the protocol would depend on what Arabidopsis tissues/effect of what treatments you are interested in. For example, you can use DCF for quantitative determination of ROS production in excised leaves by measuring the fluorescence of the incubation medium - Umbach et al. 2012 in PloS One. Hope it helps:)
Hi Lauren! You can also try Nitrotetrazolium Blue staining (NBT), to detect O2- spots (superoxide anion). Another measurements can provide you a lot of information, like catalasa activity, SOD, POD or NADH oxidase.
Hope I´ve helped!
Hi Lauren, there is a fluorescent dye called DCF, which can be used for ROS detection. You can use it for quantitative determination of ROS production, the protocol would depend on what Arabidopsis tissues/effect of what treatments you are interested in. For example, you can use DCF for quantitative determination of ROS production in excised leaves by measuring the fluorescence of the incubation medium - Umbach et al. 2012 in PloS One. Hope it helps:)
Hi
Please refer to this paper:
Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae
http://www.plantmethods.com/content/10/1/6
Thanks everyone for your responses! They were really helpful!
Having been associated with the subject of Chemistry for the last over four and a half decades, may I dare reproduce a simple method to quantitatively determine ROS in various parts of all types of plants as follows?
{See Reference}
[J. Soil Sci. Plant Nutr. vol.13 no.4 Temuco dic. 2013 Epub 30-Oct-2013]
Most of the chemicals required as available for ready use even for those who are beginners in the subject of Chemistry.
A 1-mL aliquot of the supernatant of a fresh leaf extract was added to 0.9 mL of 65 mM PBS* (pH 7.8) and 0.1 mL of 10 mM hydroxyl ammonium chloride. The reaction was incubated at 25°C for 35 min. Solution (0.5 mL) from above reaction mixture described above was then added to 0.5 mL of 17 mM sulfonic acid and 0.5 mL of 7.8 mM a-naphthylamine solution. After a 20-min reaction, 2 mL of ether was added and mixed well. The solution was centrifuged at 1500 g at 4 C for 5 min.
The absorbance of the pink supernatant was measured at 530 nm with a spectrophotometer. The absorbance values were calibrated to a standard curve generated with known concentrations of HNO2.
*Phosphate Buffer Saline.
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