Through MTT assay, i observed that a drug is causing cell death. Now to confirm the cell death pathway, i am expanding my study. I want to check the ROS generation using DCFDA as indicator. In MTT assay i treated cells for 24 hour with the drug. So please suggest me the suitable protocol for DCFDA staining. The method i adopt is:
1. Culture the cell with a desired cell density.
2. When cells grow appropriately, treat them with the drug for 24 hours or specified time.
3. After the specified time, remove the media (having drug) and add fresh media with DCFDA 1-10uM. Incubate for 30 minutes at 37°C.
4. Remove media again, wash twice with Warm PBS.
5. Add little amount of PBS for preventing the cells from drying. Then analyse under fluorescence microscope.
Is this Protocol right? Some researchers treat the cells with the drug, and at the same time add DCFDA, then incubate for 30 min at 37°C, after that repeat the steps 4,5 as above. What about this? They are on the opinion that drug causes ROS generation at once as it is added to the cells. If it is so, the it means that in MTT assay, drug should cause cell death after 30 minutes only.
Please share your comments here. Also share protocols for DAPI, Rhodamine 123, PI staining by using florescence microscope.
Dear Salman,
I also added DCFDA just AFTER drug or stimulus removal when I tested metals on neutrophils. Two points: 1) do not grow cells to 100% but at 70-80% confluency, 2) ROS production is rather an early event after a stimulus. Therefore, do not keep your substance on cells for a long time, but only for the time indicated (if any) for that drug.
Dear Salman,
In our lab, they use both method, depending on the time of your drug to induce ROS, so if your drug induce ROS in time less than 6hr, u can stain the cell at the time of drug treatment. While if your drug induce ROS in more than 6hr, so u can treat your cell first and then stain your cell to study the effect of your drug in ROS production.
hope it is helpful, Good luck.
Dear Salman
In our experiments we have employed various methods for quantifying ROS activity using fluorescence probes as well as ROS staining using various probes such as DCFDA ,DHE.etc. We have worked on various types of cells including fibroblasts, neurons as well as cancer cells among others. We found that modifications on the conventional methods available may need to be exercised due to the time variations in ROS generation as well as binding characteristics of individual cell lines if you are performing staining studies. Generally we employ the following layout:
1.culture semi confluent cells till attached (if using adherent cells)
2. treat with insult (H202, serum deprivation, mitochondrial toxins etc. for 24 hours)
3. if not performing a pre-treatment protocol then drug or antioxidant can be included directly (after about 30 mins) in wells treated with pro-oxidant compounds
4. we usually perform a time dependent ROS generation to study the effect of the antioxidant of interest, therefor the fluorescent probe can be added at the point of quantification i.e. 30 mins , 1 hr etc.
5. if doing a one off measurement, then the fluorescent probed should be added 10-30 mins before washing off (again this has to be optimized for your cell type)
6. once washed off ( several times depending on cell type) direct reading using a fluorescent plate reader for relative intensities measurement can be computed
note: for insults like serum deprivation often 48 hours is required to generate sufficient ROS, H202, 30 mins to 1 hour, mitochondrial toxins (rotenone) 30 mins to 1 hr
Best wishes
What concentration of H2O2 is used to generate ROS as positive control?
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