I did COMET assay in neutral condition. When I revealed cells with SYBR Gold I could observe this kind of black dot in the nucleus (see picture attached). Because I can't analyse with a software all these cells, I try to understand what is it and how avoid it.
It come from a contamination in the cells? in the SYBR gold? Is it apoptotic vesicule? Other suggestion?
I never saw that kind of dot when I was performed alkalin COMET assay.
Thanks by advance
Dear Hermance Beaud
Please wash the gels several times with ultrapure water and stain again with propidium iodine and check the nucleoids.
some advice is to filter the dye with 0.2 microns before each stain.
Also check is the black dots are fluorencent in another channel and after with transmitted ligth could be a chemical contamination artifact.
Regards, Jorge
Dear Hermance Beaud,
It looks like case of contamination. In which type of cell are you working on ?
Thanks for the advice Jorge. Unfortunately, ddH2O wash didn't work. I could see a little the cells with a TRITC filter (I used SYBR Gold so I have observed the cells with FITC filter), and the black dots are still dark so they are not fluorescent.
Samrat: There are not contamination in the others sample processed in the same time, so I'm septic with this hypothesis (even if I thought of it). I use a spermatogonia cell line. The culture of the cell line is free of contamination.
the black dot look like due to contamination. please carefully wash the gel and also use the ultra pure staining. if problem persistence then change the stain and use the other stain like silver EtBr. use the comet assay IV software for comet
Dear Hermance Beaud,
I believe that should change the stain and use ethidium bromide (EtBr). Regards
Dear Hermance Beaud,
Yeah you should give a try with Ethidium bromide.
Ethidium Bromide (EtBr; 10X Stock - 20 μg/mL): add 10 mg in 50mL dH2O, store at room temperature. For 1X stock - mix 1 mL with 9 mL dH2O. Use 1X stock for staining. 80µl of 1X stock is enough for staining one slide.
All the Best!!!!
With Regards,
Samrat Das
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