Hi!
I am currently optimizing a protocol for the comet assay in my lab and I was wondering if I could use a regular microscope slide for the assay? I tried coating the slide with one layer of 1% NMP agarose but I do not see the layer. Should I double coat?
Also, since they are regular slides, I do not have the circle shaped wells on them to drop my cells. Once I add approximately 75 ul of sample mixed with LMP, should I spread the drop or leave it as it is?
Any comments and suggestions would be appreciated!
Thank you !
Respected Kulbay,
Yes, you can use regular microscopic slides for the Comet Assay. Make sure that it is thin and transparent. The Agarose gel layer on the slide should also be thin when you embed the cells on it. You need atleast a digital fluorescence microscope with 400x to 1000x magnification. Believe me, it is very very simple assay and serves as bridge between cell biology and molecular biology. Good luck with your effort.
Respectfully,
Dean S. Sharma
Yes, normal microscope slides work well. You can even try slightly scratching the slide with a glass cutter pen to make it's surface rough so that, it could tightly hold NMP agarose on which the cell suspension mixed with LMP agarose is layered. Thin layer of agarose is preferable. I also prefer spreading the mix on NMP agarose rather than leaving the drop as such.
Good luck!
Hello Merve Kulbay
You can use the regular microscope slide for comet assay. After coating the slide with 1% NMP agarose you will find a very thin coat on the slide. It is normal. You need not have circle shaped wells on the slide. You can add 75 ul of sample mixed with LMP as a drop and then place a slide coverslip on the drop gradually so that no air bubbles are trapped within. Wait for the LMP to solidify for 10 minutes. Then you can use a pointed forceps or a fine needle to remove the coverslip from the layer of LMP having the cells beneath. The coverslip detaches from LMP with ease. But do this process slowly to avoid the LMP from sticking to the coverslip. This process is not difficult. Then continue with the further steps.
Best Wishes.
Regular microscope slides are fine but they should be thin and very clean and dry.The NMPA coating is not visible after drying
Thank you so much everyone for your answers! These answer every aspect of my questions. I will do an assay next week with every suggestions and let you know what went better in my lab/experimental conditions!
Goodluck to everyone with your respective research. Thank you again!
Hi Merve,
Yes you can use normal microscope slide but I prepared using frosted ones where I initially have to charge the surface by sliding one end over the surface of another. it help in allowing the NMP agar to spread evenly and gives a good coating layer.
Thank you
All the best for your work.
Perfectly clean and dry ordinary thin microscope slides previously coated with 1percent normal melting point agarose will do perfectly well for comet assay. We used them for the assessment of DNA damage in lymphocytes of suspected cervical cancer patients and the toxicity of tobacco extract on normal lymphocytes
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