how to measure ROS in cell wiht H2DCFH - (Feb/13/2010 )

I have done many times with H2DCFH to measure ROS production, but the fluorescence is very weak.

I cultured SH-SY5Y cell, then cells were treated with H2O2 to induce oxidative stress. Then add H2DCFH to measure ROS fluorescence.

I will appreciate tht the expert tell me the success protocol.

Hi,

try the following.

Load cells with DCFDA (try 5, 10, 50 and µM) for 30 min
Dilute the DCFDA in HBS
Add H2O2 or pyocyanin to one sample

Then it depends on how you like to analyse. You can lyse your cells and measure the flourescence in a microplate reader. If doing so, chack protein concentration in advance and use equal amounts. Or you can analyse your cells by FACS. If you have any questions let me know.

Cheers
Alexander

P.S. Do all incubations in the dark

Thank Alexander for your advice.

I try again with pre-loading DCFH in different concentrations for 30 min. I use fluorescence microscope to observe the glass slides.
but it is still very weak like before.
If you have done this experiment, could you please give me your detail protocol?

Thanks again.

Hi,
Ive been trying to do the ROS detection assay using H2DCFDA and cm=H2DCFDA. But so far not been successful. Iwant to use flowcytometry for ROS detection after treating cells with H2O2.
But the untreated and treated cells both have similar MFI in flow cytometry.
Please let me know a good protocol if you have been successful in ROS detection asay . I use WEhi231 a suspension cell line.
Thank you.

I would like to know regarding using DCFDA for determination of ROS

I work with HepG2 cells.The details of the porotocol I used

Seeded HepG2 at a density of 15,000 per well in 96 well black plate

24hrs post seeding, pretreated the cells with plant extracted in different concentrations (dmso) for 4hrs in complete DMEM

The MEDIA was removed, washed with KRB and hydrogen peroxide was added at a concentration of 1nM for 15min in dark at 37 degree celsius with 5 percent carbondioxide

The KRB was removed and DCFDA at a final concentration of 20micromolar (20mM stock in DMSO, WS prepared in KRB) for 20min in dark at 37 degree celsius with 5 percent carbondioxide

It was washed twice with KRB and read on plate reader

My problem is that the cells are geting washed out. Should I need to DCFDA before hydrogen peroxide? Can someone explain the reason behind it?
Can anypone send me the detailed protocol