I use DCFDCA

I use DCFDCA ( 5 microM final concentration). I use two more dyes CellROX which stain cytoplasm and DNA ( Orange and green dye).

I would add your nanoparticles/treatment as you're currently doing, but add your dye about 10-15 minutes prior to taking each measurement. ROS are incredibly short-lived, and the incubation times for CellROX, MitoSOX, etc,etc are usually only a few minutes. 

thank you so much Rodrigo..

Thank you Rebecca. Do u mean I make one plate for all time points and add dye before 10-15 mins and measure the fluorescence with media and nanoparticles in the wells, no need for washing the cells with PBS? 

because what happens is if i wash the cells, due to the nanoparticles exposed, the cells die and they are gonna be washed away. so the fluroscence i get from a well which had NPs which are toxic  will be very less compared to a well which had NPs less toxic due to the number of cells. 

If i make one plate for all time points, can I keep adding dye at different time points to the same plate and same well?

and also is there any specific temperature for measuring the intensity of fluorescence? I read in a paper which said 37 degree celcius 

As far as I know, you have to remove the media of the cells and  add DCFDCA in the darkness  for 15 min at 37 º. Once you do it, remove the dye and add the media with your concentration of nanoparticles and start to read it.

Please note, that the dye you use may overload your cells, when you do not wash your cells. That could massively affect your results.

Luminol enhanced ROS detection can be performed with a microtiterplate luminometer (LUmo).