Here's what's available in RG that I think can be helpful to your question.

Best regards,

Debra

https://www.researchgate.net/search.Search.html?type=publication&query=measure%20ROS%20using%20DCFDA&previous=publication

Thanks Debra, the protocol I initially posted is based on what you've linked me to. It doesn't seem to be working in my hands though and I was hoping that someone might be able to spot something that I haven't.

Hi Aleksey,

Thanks for that. I think you may have misunderstood my initial post. I'm not trying to measure kinetics. I tried two protocols:

Preincubate with DCFDA 45min, stimulate 2h, read

OR

Stimulate 2h, incubate with DCFDA 15min, read

I've also mentioned the concentration of DCFDA I used: 1mM which the cells tolerated well. All handling was done in the dark and incubations were at 37 degrees.

I tried FACS today as well (second protocol) and didn't see anything. I'm using NAC as a negative control and have control cells as well.

Hi Aleksey,

I'm looking at ROS production after exposure to particulate matter from pollution. I've looked at a few papers from people studying the same process using the same type of particulate matter which is where the 50uM-1mM (I tried the 50uM today with the FACS exp) and 2h (we use 2h for all our other work as well so this is our time point of interest) have come from though some people preincubate and some people add the dye after which is why I tried both. With the plate reader format the florescence readings are 1 for everything. With the FACS format the florescence readings are the same for control, stimulated and stimulated + NAC. I haven't looked at unstained cells but will include when I do this next. I'm just not sure what to try next!

Hello Reetika,

I don't want to dash your hopes but it is not recommended to use DCF /DCFDA for measuring oxidative stress (please refer to the attached publication).

Regards, Christian

Article What does the commonly used DCF test for oxidative stress really show?

Anything else you'd recommend then Christian?

I am quite skeptical when it comes to using fluorescent dyes for measuring oxidative stress. I feel that applying EPR ('Electron Paramagnetic Resonance') is much more accurate and reliable. However, you'll need somebody who can provide you with the means for conducting these measurements. 

Good luck!

Regards, Christian

Article A Quantitative Method to Monitor Reactive Oxygen Species Pro...

Aleksey, just thought I'd let you know that the 2uM with FACS seems to have worked. Many thanks

Trouble with DCF is that it also measures RNS, such as ONOO, so your signal may also vary depending on how much NO is present and not just ROS level. 

Hi everybody,

Protocol for ROS detection using DCFDA also but with adherent (BV-2 cells) for different time exposure with a Pesticide using a 96 well plate.

Could you please give me a protocol with more details to follow?

Thanks in advance

The calcium and magnesium can affect the intracellular ROS using DCFH-DA. Please read my paper titled "Influences of Calcium and Magnesium Ions on Cellular Antioxidant Activity (CAA) Determination". I hope it is useful for you.

Article Influences of Calcium and Magnesium Ions on Cellular Antioxi...

Hi Reetika,

Can you share how you resolved the problem?

Thanks