I want to check the production of ROS by using DCF-DA as indicator in Colon cancer cells. What is the optimum time after which a drug starts the generation of ROS? 30 min, 1 hr, 6 hr 12 hrs, 24 hrs? Does anyone know? I mean that for how much time i should treat the cells by the test drug?
ROS generation is considered as a rapid process. However, different cell types could respond with a different manner. In my opinion, you could treat your cells with the respective concentration of drug for different period of time (e.g. 30, 60 and 120 min) before proceeding to the measurement of ROS generation. Afterwards, you will have an idea of cells' response as well as drug-mediated ROS generation.
It depends upon the drug and also the cells which you are using to some extent. So better do time dependent study to check the initiation point for ROS.
Here is a protocol: they recommend 30 to 60 min. Check if that fits to your concentrations.
http://www.cellbiolabs.com/sites/default/files/STA-342-ROS-assay-kit.pdf
Hi, First of all i want to know whether u were using a light sensitive drug or non-light sensitive drug?
I will give you in the idea of using a light sensitive drug, (i.e. for photodynamic therapy). After treating the cells with light sensitive drug, give incubation time of 4hrs for the cells to uptake the drug and give a wash with PBS. Then replace with the fresh medium and give light treatment for the period of which u required and finally incubate with the ROS marker and again wash with PBS after the time period u want to watch the cells using Phase contrast microscopy..
I hope this might help you...
Plz check my article......may help.........
http://www.ncbi.nlm.nih.gov/pubmed/23876794
Typically people grow cells in >10 mM glucose and >220 micromolar oxygen; so that the autooxidation of the reducing sugar glucose is the largest single source of superoxide/hydrogen peroxide.
One cannot, a prior, know if an increase in DCF generation in response to an effector is due to an increase in ROS generation, in the contect of a H2-DCF assay. The rate of oxidation of the probe is a function of two rates; ROS generation and ROS detoxification by (mostly0 enzymatic antioxidant pathways.
If add 'X' to cells causes a rise in the DCF signal then 'X' coul be increasing ROS generation, dropping antioxidant capacity or both.
Wash cells. Add H2-DCF-AM and place in pre-warmed plate reader. Examine the rate that DCF is being formed. After 30 minutes add 100um-1 mM H2O2 and examine the rate of induced H2-DCF oxidation.
Remove supernatent, add H2O2 and read on same plate reader settings to get a calibration.
The ratio of the uninduced/induced rates will tell you if you have increased ROS generation or attacked an antioxidant pathway.
Now add hoechst, wait, and fix cells. Count cells and carefully caltulate DCF rates PER CELL.
Dear Salman,
Can you tell me the drug you are testing and the cells/animal on whom its is tested. The ROS production is subjective matter and I can help you more if you can provide me more information.
All the best
Regards
Jitendra
Salman,
If your cells are adherent, I have had success using a fluorescent plate reader to measure ROS using DCF-DA. After incubation with DCF-DA (30 min, for example), various compounds and concentrations of test chemicals can be added and fluorescence can be measured over time simply by repeated measurement of the plate (of course, placing the plate back in the incubator to maintain CO2 and temperature). Time-points of interest can then be validated after you have an idea of the timing. I found that some chemicals cause ROS increases beginning at 4 hours and this increased over 24 hours, but this is obviously compound- and concentration- dependent. I hope this helps
Best of luck
ROS production depends upon the time period for drug treatment, cell types used and drug concentration.
It would seem that running a time course will answer that question for you. Since ROS generation occurs quickly following various treatments/stressors, I would probably recommend measuring a time point before 30 mins. We measure H2O2 production using luminol in UV-irradiated skin cells and we start the measurements at 0, 15m, 30m, 45m, etc.
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