I have been using mouse mammary adenocarcinoma cells and I have recently observed black dots. I was doing Puromycin-kill curve and so the cells were treated with Puromycin. I am not sure if the black dots are cell debris or mycoplasma. I also have untreated one where the black dots are very less in number compared to the treated ones. Been going through lot of related questions.. I would say they are irregular in shape, not moving and are adhered. I have not done DAPI yet to check for mycoplasma..
I think it comes from cellular debris, i inform you that the recommended Puromycin dose for selection of cell cultures is of the order of 1 to 10 µg/ml. Although it can be toxic to eukaryotic cells at concentrations as low as 1 µg/ml. It works quickly and kills up to 99% of non resistant cells in less than two days (Reference Wikipedia). Some Mycoplasma strains are sensitive to the action of Puromycin look at the link below
Good luck
http://www.ncbi.nlm.nih.gov/pubmed/4321093
Hi.. Thanks for the answer. Yes, my supervisor also suggested the same thing but surprisingly the cells I am using are pretty resistant to Puromycin and it required 7 days for all the non-resistant cells to die. I am performing the experiment again but this time I will be looking for Puromycin effect two days after treatment and hopefully all the non-resistant cells will be dead. With respect to the contamination, the plate from which I re-seeded the cells to 24-well, that plate was positive mycoplasma contamination. But I guess the 24-well plate that was treated with Puromycin should have killed the mycoplasma based on the paper you referred. Thanks for the paper!! It was a good one..
Hi Patrick!! I am using DMEM medium.. Does usage of different medium have any influence?
I am actually trying to understand your response.. You mean to say that the formation of meladonin is the reason why I see black dots? And meladonin acts as a photo-sensitiser?? Am sorry if I misunderstood your response.. But this is totally a different view point I never thought of.. And does the concentration of mercaptoethanol and :-cysteine matter? Like how much should I add??
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