Hello
i use h2O2 (100uM) for 24 hrs to mimic ischemia model in the cells (cardiomyocytes) . so when i do ROS measurement using DCFH-DA, do i need to add a positive control like ROSUP(solarbio kit) or h2o2 again in my positive control sample? because i have already treated the cells with h2o2 for 24hrs.
(i will use confocal microscope)
please comment.
Hi Syeda, The differences you see in DCFH-DA between your negative and positive control can be very small by flow cytometry (500 channels on Becton Dickinson LSRII) so I doubt in my experience you would be able to image this before photobleaching would spoil any chances of you getting meaningful results
Regards
Gary
I have published 2 papers using DCF to detect ROS that might help you.
The first, a very simple one. Using an old fluorescent microscope, was done on a cell line I "created" that had high ROS production and it was possible to detect the difference. I still remember the wonder of seing the result so clearly. So I disagree with Dr Warnes answer, you should be able to detect the difference using a microscope.
Article Oxidative Stress and Upregulation of Mitochondrial Biogenesi...
The second paper uses a fluorescent plate reader to measure ROS in cells growing on 96 well plate which allowed me to test many different conditions. I only treated the cells with 500 uM H2O2 for 30 min before the measurement. This are HUVEC cells, also primary cultures. It is always nice to have a good positive control. I suggest you try at least in your first assay to have such a control in an extra well (or plate). I also found that the best positive control was to live the cells out of the incubator at RT for a few minutes. This stress increases ROS production very fast.
Article Juice and Phenolic Fractions of the Berry Aristotelia chilen...
Conclusion: I recommend you include one or 2 more positive controls. And of course also 1 or 2 negative controls. You should also have a control with a good antioxidant to show that you can revert the effect, this will strongly suggest that the changes you see are due to ROS increase.
@all thank you for your suggestions.
@Dr. miranda, in which plate you seed your cells and at which density? can i use 24 well plates and then visualize under fluorescence microscope?
i recently used confocal specific small culture dishes to seed cells (primary cardiomyocytes).. did ROS ASSAY. and when i checked under confocal, there was no cell at all, except for control group. the concentration of drug and h2o2 i have already optimized uing MTT.
MY PROTOCOL WAS as follows
'' after incubation with drug and h202 , i wash cells with DMEM, then add 10uM/L of DCFH-DA, enough to cover the cells (almost 1 ml in those dishes) , incubate for 20-30 minutes and then wash three times with DMEM. Add Dmem fourth time and visulaize under confocal."
PLEASE CHECK IF THERE IS ANY MISTAKE WITH PROTOCOL?
Hello Miranda, I just saw your papers. I am a question for the detection of ROS by using the Fluorescence microscope.
If you are going to treat the cells with H2O2, you are going to treat them before or after the DCF dye?
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