I'm treating my CHO cells with 100uM H202 for positive control. cells are dissociated by 20mM EDTA in 1X PBS. Trying to do all procedure under low light. 1% Low melting agarose temperature was 37C. Lysis was done in 2% lauroyl sarcosine in 500mM EDTA with 0.5 mg/ml. over night lysis at 4C. electrophoresis was done with 21V for 25 min at 4C. But I'm having comet tail both in my positive and negative cells. Could anyone help what should I modify?
Hi,
concering your negative control: Do all cells have a similar tail, or are you referring only to a certain fraction of cells? A certain amount of cells with tails in the negative control is perfectly acceptable; those are cells in S-phase, where numerous replication strand intermediates exist, which appear as heavily damaged cells in the comet assay.
If all your cells show a significant comet, I would assume that there probably is damage done to the cell post-lysis. We generally use a Triton X-100 based lysis buffer with 10 % DMSO, which acts as a radical scavenger, protecting the naked DNA from oxidative damage, so maybe try supplementing your buffer with DMSO. Otherwise I would blame light exposure at some point between lysis and neutralization. Naked DNA is very sensitive to visible light, so try to work with red light and only if you absolutely cannot avoid it.
R your cells happy? Are they at very high density?
The reference below should be helpful
http://users.ox.ac.uk/~atdgroup/technicalnotes/Comet%20analysis.pdf
Hope it helps. Good Luck!
Hi,
concering your negative control: Do all cells have a similar tail, or are you referring only to a certain fraction of cells? A certain amount of cells with tails in the negative control is perfectly acceptable; those are cells in S-phase, where numerous replication strand intermediates exist, which appear as heavily damaged cells in the comet assay.
If all your cells show a significant comet, I would assume that there probably is damage done to the cell post-lysis. We generally use a Triton X-100 based lysis buffer with 10 % DMSO, which acts as a radical scavenger, protecting the naked DNA from oxidative damage, so maybe try supplementing your buffer with DMSO. Otherwise I would blame light exposure at some point between lysis and neutralization. Naked DNA is very sensitive to visible light, so try to work with red light and only if you absolutely cannot avoid it.
Thanks a lot Patrick. Almost all of my cells having tail in negative control. I will attach two images here. please take a look.
Hi,
I agree with Harald, try it with less voltage and current and / or shorter electrophoresis duration, those tails are definitively way too long even for a positive control. Field strengths of approx. 0.7 to 1.0 V/cm for 20-30 min are generally used, the actual voltage and current set on the power supply depends on your paricular gel chamber (distance in between the electrodes) and resistance (amount of buffer over the slides).
Optimization of electrophoresis parameters is best done with a positive control, ideally you'll want tails which are roughly 3-6 nucleoid (head) diameters long. After that, proceed to troubleshooting your negative control, however excessive voltage may contribute to or even completely explain your problem.
I strongly disagree with the statement on current. Current has no effect on the tail length, as it is a resulting factor from the voltage and the resistance of the used solution. So adjusting the voltage will do the job to reduce the tails. However, from what I see in the pictures, there is more to it than pure voltage depending conditions. Besides voltage please do also look at the temperature in the tank at the end of a run. An increase here will cause significant longer tails as well. Further, what has been you final agarose concentration in the spot? tail length is strongly correlated to this concentration
If you face issues in your negative control please do also verify your freezing and thawing procedures. The DMSO in the freezing media will cause longer tails if thawed for to long. Hope this helps. If you got any further please do not hesitate to contact me anytime.
Hello Imran,
As you mentioned the brief Protocol and attached Images, I am suspecting that Voltage used by you might be one of the contributing factor for tail in negative control. However, the use of DMSO acts as a radical scavenger, protecting the naked DNA from oxidative damage. But the use of DMSO supplemented lysis medium mostly required for cell containing cheating agents I.e. Blood ( hemoglobin in RBCs contain iron).
You need to check following
1. Reduce the voltage 0.7-.1 v/cm for 20-30 min at strictly 4 degree centigrade temperature.
2. percentage of Low melting agarose: 0.7-1 %
3. pH of Lysis Solution also contribute the Tail length
4. Researcher Generally use 1% SLS
For more you can also refer
Article Sesamol ameliorates radiation induced DNA damage in hematopo...
Hope it helps. Good Luck!
Arun
Thanks a lot to you guys for your precious opinions. I want to share two more images. These are human Breast Cancer cell line. I run them with the same procedure as previous one and the electrophoresis was done in the same tank with the CHO cells. These seems run short, is it?
Had two 60 mm dishes with BT20 cells. one is negative control, kept 37 C in the incubator until dissociated. The positive control plates were treated with 100 uM H2O2, and incubated for 20 min at 4 C. Then, I used non-enzymatic cell dissociating solution (Sigma) approx. 1 ml and kept the both plates at 37 C for 10 min. I didn't used any complete media to inactivate the solution after 10 min. Used repeated pippeting to dislodge cells from plates. and then go for cell count, followed by diluting with PBS to get desired number.
Hi,
harsh pipetting for cell separation will indeed mechanically damage your cells, resulting in DNA fragmentation. Is there any reason for you to use non-enzymatic dissociation medium? Good old trypsin will do a great job and will ideally give you a aggregate-free suspension without excessive pipetting (I guess resulting from usage of enzyme-less dissociation medium). This is especially important if you dissociate primary tissues for comet analysis, see also this article: Article A novel contact assay for testing genotoxicity of chemicals ...
This may an explanation for the damage in your negative control.
Concering your latest images: I have seen a similar "smear" in slides without cover layer over the cells; many protocols omit the third protective layer over the cells ("sandwich"-principle, cell-free bottom, cell-containing mid and another cell-free top layer of agarose), which I personally found to be not a good idea. Can you give more detail on your slide preparation process (esp. number of layers and spreading technique)?
I found somewhere in research gate that problem of high comet in negtive control may be caused by trypsin, that's why I switched to non ezymatic solution. I also used 20 mM in 1X PBS to dissociate cells. As you see both have the same outcome as trypsin.
Details of slide preparation:
After getting a desired cell number, I took 50 ul of 1% LM agarose (at 37 C) and 450 ul of cell suspension (in PBS, on ice) and mix by pippete. And spread on Trivigen comet slide. The slides are not pre-coated and I did not use any additional coat on top of the cell's layer. (Trivigen comet assay protocol said to use this way) I kept the slide at 4 C for about 20 min, then go for lysis.
Hi, you can also try to reduce lysis time from overnight to 1h lysis. It will not completely solve your problem but the damage will be lesser, also reduce voltage to 0.7 V/cm for 20 min, preparation of sandwich with additional layer of agarose over the layer with cells should also help...
Does anyone think that Sk. Imran Ali's cells (original pair of photos) are apoptotic?
- Badges
- Science topic