Each time I do the comet assay, I get variation. It works just one time for each five I do, even if I am using the same vial of the damage agent, or the same lysis buffer or the same electrophoresis buffer. The last times what I've gotten is that using the same time of incubations, all my cells are damaged as hedgehog, even the control ones, or well, I don't get damage at all.
The first picture is from my control cells, and as you can see, every single one of them is damaged. The second one is from the cells I exposed to Irinotecan (campthothecin that generates single strand breaks) 0.1 mM that are as well as my control cells (the ones that are not hedgehog). Both of them had the electrophoresis at the same time and were treated for the same time with the unwinding buffer.
The protocol I use is the next one:
I get the cell by tripsinizing them for 5 minutes and then I spin them and resuspend the cell pellet in 60 µL of PBS.
Then, I mix 7.5 µL of the cell suspension in 45 µL of 0.75% LMPA, and I drop this solution on a 1% NMPA precoated slide. Then, I carefully put the coverslip on the cell solution and I incubate it at 4°C for 2 minutes. Then I remove the coverslip and the slide is incubated for 1 hour at 4°C with lysis buffer (3.72g EDTA, 14.6g NaCl, 1mL Triton X-100, 0.12g Trizma base in 100 mL of water, pH=10).
Then, I incubate with electrophoresis or unwinding buffer (300 mM NaOH, 1 mM EDTA) for 40 minutes, and after that I put the slides on a horizontal electrophoresis camera with 400 mL of electrophoresis buffer (3 mL of buffer above the slide) and I run it at 25 V (260-310 mA) for 20 minutes.
Finally, I do three washes with neutralization buffer (7.88g TrizmaHCl in 100 mL of water).
I did the same for both of the pictures and I got those different results in the same run, so I don't understand what am I doing wrong. I have done previously the same experiment and it has gone well but the last month it has been like this. For coating the slides, I submerge the slide in a 1% NMPA solution that is hot, and then I let it cool at room temperature until it's dried.
In this experiment, I used MEF that were at a confluency of 60-70%.
Hello Zaida Escila Martínez Moreno
I want to know the kind of cells that you used (damaged cell or normal cell) and if you worked at obscurity conditions
Hello Himri Imane
I use Mouse Embryonic Fibroblasts, for both treatments. I just made a comet assay yesterday and I got the same results; half of the slides are totally damaged and the cells that are suposed to be damaged are almost as well as the control. I work all the time at obscurity conditions and yesterday I added all my solutions at 4°C (I used to add them at room temperature always).
Hello Zaida
Try to reduce the time of electrophoresis "electrophoresis alkaline buffer (300 Mm NaOH and 1 mM EDTA, pH > 13) for 20 min at 4 °C. Electrophoresis was conducted at 4 ° C for 20 min at 25 V and 300 mA". see my article
good luck
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Thank you! But I already do that, my electrophoresis time is 20 mminutes at 25V and 300 mA, the only difference is that I don't do it at 4ºC; but the last time what I did was to cool the electrophoresis buffer at 4ºC though the electrophoresis was done at room temperature. This way it didn't work, maybe I will try to do all the electrophoresis at 4ªC.
Harald Kühnel, I have tried neutral conditions but I do not observe any damage, because I'm looking for single strand breaks. My slides are always protected from light. I did it at cold conditions last time and it didn't work again, so I don't know what's happening. A mexican professor told me I should've used the NMPAgarose disssolved in PBS, so I also did that and in fact, I didn't see that many phantoms (hedgehogs), but they were still there. The problem was that I didn't see any difference between my control and my damaged cells...
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