I am working on HEK293 cell line. The cells were treated with 5-FU for 24hr then I performed comet assay. Visibly many cells were dead but when I did comet assay nothing was observed (No comet). No intact nuclei. Some kind of ruptured nuclei was seen. I have incubated slides in lysis buffer for overnight. My elecrophoretic run was more than 50min. (Recommended run time 30 min). Can anybody suggest some remedies? Should I try less treatment with 5-FU than the 24hr?
It is recommended treating the cells for 6 hours since DNA damage could be repaired in 24 hours. But as you desdribed cells are dying in 24-hour treatment. Maybe your positive control does not have genotoxicity-related mechanism of action, which is very unlikely. Try using higher concentrations or aplying other drugs. For example: I used doxorubicin at 2 uM and the DNA damage was very high on U-343 and MCF-7 cell lines.
Your lysis method is perfect. What eletrophoretic buffer are you using?
Best regards.
I think there must be something scavenging your drug effeciency. Have you tried to set a positive control ( Simply treat yourt cells with 50 uM H2O2) for 30 min/on ice/ protected from light, then run your experiment?....hope this work with you
Regards
Hishyar Najeeb
I am using 5-fluorouracil which very well known anticancer drug. And i have also read some articles regarding DNA damage. It causes apoptosis as well as single stranded DNA damage to the cells. I feel the positive control is good. But the major point of concern is eletrophoretic mobility. I am using alkaline comet assay. So my buffer is mixture of NaOH and EDTA. I feel that time point as u said is very important. I should try with one hour or four treatment. Thank you for suggestions.
H2O2 is the best agent for setting a positive control. This will tell you that the experiment is working. Another point about your assay, is the repair mechanism, as you know SSBs are very easy for repairing. try increase the dose/time of exposure.........then keep your sample on ice after treatment to avoid further repair.
Hey,
from my experience, dependent on the exact genotoxicity mechanism of the respective positive control, a few show a peak response, which will result in decreased tails when concentrations are increased. We observed this for UV-irradiation and a few (alkylating) chemicals, so reducing your concentrations or exposure duration may help.
As Luis has already pointed out, the genotoxic mechanism of 5-FU may not cause enough damage in your exposure scenario, as it is a base analogon, which requires incorporation into DNA to be effective, which is only the case if DNA replication or a significant amount of excision repair takes place. We usually stick to UV irradiation using a crosslinker, which is a simple, quick and reproducible way for a positive control exposure. H2O2 also works well, but be sure to carefully test adequate concentrations and exposure durations, as excessive damage (which may occur at concentrations as low as 0.1 %, dependent on the cell line) will totally break down your DNA, producing only diffuse clouds which can not be visually analyzed after staining.
Dear Piyush,
I worked with cell lines long time ago but to my mind even 2 to 4hrs would be enough to see comet tails. Even in vivo on mouse model we able to see the response in our positive control after 2 and 4 hrs.
Thank you all for your valuable answers. This is going to be help me a lot. Thanks again.
Dear Piyush, I think you must run a positive control using H2O2 first to see whether your comet assay standard is OK.
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