I have a HNSCC cell line resistant to a drug (2 different clones) showing different morphology than the parental cell line. Attached are phase contrast images of these cells. There are black dot like structures visible, within the cytoplasm (probably aggregates, but of what?). Anyone seen anything like this in their experiments. I suspect this maybe the reason for unusual results in my subsequent experiments. Can this be eliminated in anyway? How can i know what it is exactly? Any thoughts and inputs will be highly appreciated. Thanks in advance.
Hi Pooja,
I just saw your question, it is a interesting one. Because you have different clones my best guess is that black dots on your cells are in correlation with the genotype of your clones. In order to prove this point, you will need to perform rescue experiment. On KO cell line of your gene you introduce your gene and then observe what is happening with your black dots. If they disappear after rescue they are indeed an phenotype that is correlating with your genotype. I hope you did not change medium or you did not add different drugs on your mutant cells in respect with parental cell line. The other point that comes to my mind is construction of cell lines. Did you make stable or transient cell line? If you made stable cell line sometimes if you added too much of selection antibiotics it could happen that cells don’t look good (is these case cells look rather sickly). Maintaining high levels of selection antibiotics in stable cell lines sometimes is not so good long term strategy.
On first question if there is correlation between genotype and black dots, I guess you can’t remove them.
On second question what are they exactly I would go and think about function of my gene and there should be an answer. You can’t really answer the question what are those structures without immunocytochemistry. Otherwise is just guessing game.
On third question does this structure have influence on subsequent results…it is difficult to say without answering to the previous points. If you performed all correct controls....then no.
I hope I gave you some material to think about, or maybe I will start the discussion on the topic.
Good luck
Nikola
Hi Nikola,
Thank you for taking out the time to respond to my query. I would like to clarify that these cell lines in question are not different in their genotype. The clones are as a result of treating them with drugs to obtain an acquired resistant cell line. Therefore, I have parental cells which are sensitive and two monoclonal populations that are resistant to drugs. So, they have been maintained in relatively high concentrations of drug for a long period of time (as that is the selection pressure for acquired resistant cell lines)
On a different note, I completely agree with your explanation if I had a difference of genotype between these cell lines. P.S. I have fingerprinted these cell lines so, yeah they are not different.
Shoot me with your thoughts about this!
Thank you
Pooja
Hi Pooja,
I have some experience with induction of resistance in cancer cells.
Recently i induce resistance on breast cancer cells. I got three resistant clones from the same cell line and i'm not sure if all of them, but i'm sure i've seen those little dots on my resistant clones.
I´m not sure what they could be, but i don't remember getting weird results when seeing these dots. My guess would be that those are drug aggregates (inactivated drug precipitates), since your resistant cell lines are constantly growing in the presence of drug (another guess, since that's how resistant clones are "preserved"). Also, the difference in morphology could also be due to the stressful conditions in which your cells thrive. In my case, resistant cells have very similar morphology to the parental cell lines when grown without treatment. Once i culture them in the presence of drug, their morphology changes and stays the same until drug retrieval.
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