Dear all,
I'm facing a problem in solving the next points in comet assay:
1. What is the recommended media or solvent to prepare dilutions of H2O2?
2. Is it necessary to centrifuge the mixture (blood + H2O2) after incubation and discard the treatment matter? And if yes, is it PBS that will be used then to resuspend the blood? Or directly adding LMPA?.
3. When I run electrophoresis, I set amperage on 300mA, but the value displayed on the power supply doesn't reach that point (240 mA maximum). Does that make a significant difference in results?
5. What is the best concentration of Ethidium bromide to be used for staining? what is the recommended amount to be added to the slide? And What is the duration of staining?
I really in a big need for reliable answers made up on your own experiences.
Many thanks in advance.
Hi Mohammad, I will help you as much as I can.
1. We made H2O2 up in water and added directly to the 6 well plates etc.
2, I'm not sure as I only worked with Caco-2 cells. I do know that H2O2 will need to removed to prevent excessive damage. I exposed my cells to H2O2 (50 μM) for 30 minutes, then I neutralised it using FBS.
3. Electrophoresis was carried out for 25 mins at 4°C with a current of 25 V (300 mA).
4. My slides were stained with ethidium bromide (20 μg/mL). I add a few drops from a dropper, enough to fully cover the mini-gel. I leave the stain on for 10 minutes, then wash the slides thoroughly (yet gently) using water.
The alkaline comet assay performed is as follows; 100µl of blood sample was mixed with 200µl of 2% low melting temperature agarose at 37°C and then placed on a slide pre-coated with a thin layer of 0.5% normal melting agarose. The cell suspension was immediately covered with a cover glass to obtain a uniform layer, and the slides were kept at 4°C for five min. for solidification The coverslip was removed and a final layer of 0.5% Low Melting Point (LMP) agarose (100μl) was placed on the slide and covered with a coverslip.
100µl of blood sample was mixed with 200µl of 2% low melting temperature agarose at 37°C and then placed on a slide pre-coated with a thin layer of 0.5% normal melting agarose. The cell suspension was immediately covered with a cover glass to obtain a uniform layer, and the slides were kept at 4°C for five min. for solidification. The coverslip was removed, and a final layer of 0.5% Low Melting Point (LMP) agarose (100μl) was placed on the slide and covered with a cover slip.
After removing the cover glass, the cells were lysed in a lysing solution (2.5M NaCl, 100mM EDTA, 10mM Tris, 1% Triton X-100, pH 10) for one hour. After washing with redistilled water, the slides were placed in a horizontal gel electrophoresis chamber. The chamber was filled with cold electrophoretic buffer (1mM EDTA, 300mM NaOH, pH 13) and the slides were kept at 4°C for 40 min to allow the DNA to unwind. The electrophoresis was performed for 25 min (1 V/cm, 300mA) and the slides were washed three times with neutralization buffer (0.4M Tris, pH 7.5). All preparative steps were conducted in yellow light to prevent occurrence of additional DNA damage
The slides were stained with ethidium bromide, air-dried and immersed for five minutes in cold water and stained for five minutes with 80μl EtBr (20μg/ml) The slides were rinsed in cold water to remove excess stain and covered with a coverslip and analyzed with a fluorescence microscope (NIKON Eclipse 400) equipped with a CCD-4230A video camera. For EtBr, a BP 546/10nm excitation filter, and a 590nm emission filter were used. For each slide, 25 randomly chosen nuclei were analyzed, and three slides were evaluated per treatment with repetition. From the repeated experiments, the averaged median percentage of tail DNA was taken as the primary measure of DNA migration for each treatment group. The digital images were acquired and analyzed using the CASP software.
Human or animal? If latter which species (haven't worked with human blood)? I use Comet assay on marine invertebrate haemolyph and fish blood. In both cases we just add 100 uL blood (haemolymph dilueted 1:1 in somotically corrected HBSS; fish blood diluted 1:1000 with PBS) to 96 well plate and add the same volume of H2O2 GelRed (2uL in 10mL DH2O) works much better than EtBr and is far less toxic. See our relevant papers at http://markhartl.hw.ac.uk
Hope this helps.
Thank you so much for your helpful answers. But what about that point related to the amperage? Does using 240 mA instead of 300 make a significant difference?
1. I would recommend you to use sterile PBS as solvent. Normal water may induce osmotic stress in cells.
2. I would suggest you to centrifugate cells once to avoid the H2O2 reaction with the DNA after cell lysis. PBS would be a nice thing to resuspend in. No idea what you refer to with LMPA. If it is a lysis buffer, absolutely prohibited, as you would destroy your samples. If it is a classic medium, just make sure it is transparent so that it does not interfere with the comet assay visualisation.
3.I strongly suggest you to fix voltage rather than electrical intensity. After all DNA migration depends upon voltage and slight variations in the electrophoresis buffer would lead to changes in voltage for the same electrical intensity.
4. I have never used ethydium bromide. However you can find good suggestions between other comments.
Hope it helps.
Hi Mohammad,
If your electrophoresis bath is too large for power supply instrument you will get a less mV (despite the voltage) and the tails of comets will be shorter. You can add less buffer in the electrophoresis. It is important to cover all slides in the bath with buffer, but do not overfill. Try.
Thanks Snjezana . But my electrophoresis chamber is small; its 21cm long between electrodes. And I barley cover slides with buffer; some millimeters over the slides. Though, the amperage reading displayed on the power supply doesn't reach the entered value (300 mA).
sounds like you need to add more buffer. The amperage should increase.
I use to set the voltage and adjust the amperage with slight variations on the volume of the electrophoresys buffer. Normaly it works well.
Normaly I perform the dilution of H2O2 in MQ wather. Only the final concentration of it should be hight enought to use small amounts of it and do not stress the cells.
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