Hi, Vivek,

which type of the cells do you used? Do you want to detect intracellular ROS accumulation or extracellurar (apoplastic) one?

For the intracellular ROS accumulations you can use several fluorescent dyes.

Or DAB statining.

For the apolastic ROS your can use DCHBS/AAP method, whciuh is very sensitive and give you quantitave results.

But detailed protocol is dependent from whole experiment design. Cutured plant cells is much more easy! Good luck!

Thanks Dr. Taras, Your answer was useful. I am checking for hydrogen peroxide at real time, that is, when an insect oviposits into the plant. I would like to detect both intracellular and extra-cellular accumulation. Please guide me on this. thanks and regards.

Ni, Vivek, which type of the cells have you used? The methods may be sklightly difefrent for different cells types. Also, insect cells may produce peroxide as well, it may be the problem to distinquish. Moreiver, I thunk that it will bebetter to detect superoxide, because it is generated first.

Dear Dr. Taras, I am using the cells of fruits of Cho Cho (Sechium edule).

Thanks, Vivek, I understood. In any case you can measure ROS only in the liquid medium, so, I suggest you the folliwing steps:

1. You pre-adapt cells in the liquid medium, may be 1/2 Hoagland for few hours (4-6 is enough).

2. For the extracellular H2O2 you can use DCHBS/AAP method: (Van Gestelen et al., and you can see also description in my paper). So, you prepare stock of both compounds and add these compounds to the plates with the cells. To see really burst, you should take 1-2 time points before addtition of the bacteria, and than add bacteria and take 2-3 more time point (you need to take 0.2 ml of the medium and freeze it at -20C). The principal of the emthod is the following: DCHBS/AAP/POD in the presence of H2O2 formed red coloration product with A510 maximum, and A510 is directly proportional to the H2O2 level). By measure A510 you will quintify extracellular H2O2 level.

3. For the intracellular ROS you may use H2DCFDA dye: you add this dye (1 µM final concentration) and observed intracellulr fluoresence after 10-15 minutes incubation in the dark. In this case you can use simple epifluorescent microscop. Try to minimize sample illumination with fluoresent light, the dye may autooxidaized!

There are may other dyes, but I hope if you reach sucees woth thsi 2 method, it will give real picture!

Pleese tell me if something is not clear for you.

Good luck!

Thanks a lot Dr. Taras. Can you please send me the papers you have mentioned. Its can be sent to [email protected]. Please do the needful. Thanks and regards.