Hi!
I am performing an alkaline comet assay (CHO K1 cells) by setting a protocol based on several publications. I've followed every step and recommendations, but I still see comets in my control samples.
I've attached raw images of control vs treated samples, stained with Hoechst 33342 (we don't have DAPI in the lab nor the appropriate microscope filter for SYBR Green or ethidium bromide). I've even added DMSO and triton to the lysis buffer (1 hour or 2 hours, at 4°C) to reduce DNA breakdown during manipulation, and been very careful during trypsin dettachment.
It is not only a proportion of cells that present comets, but the majority. I can also note that the amount of DNA that not migrate towards the tail is still notorious, but incubation with stressors is for 24h. The positive control (hydrogen peroxide 100 uM) works fine.
If anyone could help me to find out why this is still happening, it would be very helpful. I can specify the major steps of the protocol or upload more images, if needed.
Thanks in advance
Hi Santiago,
The alkaline comet assay is too sensitive, you can avoid trypsinization and use Rubber policemen instead for the cell detachment. Additionally, try to perform the assay in low/ dim light to prevent light exposure, which can also cause DNA damage to the cell.
Thanks for your answer, Mr. Khozooei. My concern is that cell scraping would damage the cells more than trypsin does, as I've never used the scraper or a Rubber policemen before. Would you still recommend it for this technique?
Dim light is another interesting suggestion, and I'll try it combined with the advice about scraping depending on your answer.
Greetings,
Dear Santiago Elías Charif,
Trypsin can cause DNA damage to the cells as well, for me scrapping works better, in case you want to use trypsin, you need to reduce both the amount and time exposure to it.
Best regards,
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