hello.. please help me with this.
I do ROS analysis with microplate reader as well. i load the dye to cells after 24 hrs of their growth and wash them after 30 mins, add fresh media 1% FBS and then add the nanoparticles which I'm investigating if they cause ROS production. Then I take fluorescence of the plate at different time points up to 48 hours. I was curious to know if this is a good idea? Do the cells have the dye in them for 48 hours and when the cells grow do the new cells also have the dye in them?
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).
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