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why the ROS level in the negative control is also increasing??? - (May/04/2011 )

Hello everyone.

I'm doning experiment on intracellular ROS measurement with DCFH-DA and microplate reader (not flow cytometry). Simply, I pre-treat the Capan-1 cell-line (adherent pancreatic cancer cell-line) with my antioxidant for 24h, then load the cell with DCFH-DA, and finally measure by microplate reader.
Here is my procedure:
1. Seed the cell (1*10^4 in 200uL) in the 96well plate in triplicate.
2. Add different treatment (the H2O2 will be added before measurment), including: 1)+vehicle control-H2O2, 2)+vehicle control+H2O2, 3)+antioxidant-H2O2, 4)+antioxidant-H2O2.
3. 24h later, wash the cell with pre-warmed PBS once.
4. Load the cells with 50uM DCFH-DA in medium (without phenol red) for 30min, 37C in incubator.
5. Wash the cells with PBS twice, then add 195uL medium (without phenol red) to each well
6. Just before the measurement, add H2O2 to corresponding well to a final concentration of 500uM. Start the measurement immediately.

The problem for my experiment is that for the group without H2O2 stimulation, the ROS level is also increasing severly...

At first, I guess this is because the stress to the cells during the procedure, so I simplify the washing step (omit centrifug since the cells are adherent) and let the cells stay in the incubator for 1.5-2h after loading DCFH-DA.
But this is not helping, the ROS level is still increasing in the no H2O2 group.

Does anyone have any idea to solve my problem???
Thanks a lot!!!!!

-flyeryeung-

Can anyone help me with this problem???
HELP!

-flyeryeung-

Forgive me for ignorance, but do you have a control with no treatment at all? Can you have cells with DCFH-DA and nothing else?
If it is not a big increase it should be fine (it will never be 0 or stay the same), those ROS are so rebelious : )))

-kajmak-

kajmak on Sun May 8 00:35:10 2011 said:


Forgive me for ignorance, but do you have a control with no treatment at all? Can you have cells with DCFH-DA and nothing else?
If it is not a big increase it should be fine (it will never be 0 or stay the same), those ROS are so rebelious : )))


Thanks for your opinion :)

I have used a negative control before (just DCFH-DA, nothing else), but the fluorescence is also increasing...

-flyeryeung-

ROS are present in the air as well, particularly superoxide. When there is superoxide there is hydrogen peroxide, which is very reactive. Maybe that is why you are observing an increase with the dye only.

-kajmak-

kajmak on Mon May 9 12:23:43 2011 said:


ROS are present in the air as well, particularly superoxide. When there is superoxide there is hydrogen peroxide, which is very reactive. Maybe that is why you are observing an increase with the dye only.


I don't think this is the reason...
Still, I guess the stress to the cell during all the procedures cause the rising of the ROS...

-flyeryeung-