Hello! My SY5Y cells shapes were normal when firstly received(Figure 1), but changed into something like amoeba after digesting with pancreatin/EDTA for merely 30s(Figure 2, red arrow). The cells still multiply after shapes changed, but seems to be more easily to form into clumps and float. It makes me hard to perform experiment like phase contrast shooting. Does anyone know the reason? Or ever encoutered such case once? Appreciate for any suggestion.
Hello Andreas Alcatraz ,
I am not at all familiar with pancreatin-EDTA, do you use it like trypsin-EDTA to detach your cells from your surface ?
In any case, your cells seem to have been stressed by something ; I would also worry about the "cloud" in the middle of your second picture, is it only round cells in suspension or is there something else ? If the culture is contaminated, it might explain at least partially why your cells have a different morphology than before.
Do your cells grow as before, or do they take more time to reach confluence ? I would suggest that they did not really appreciate the treatment with pancreatin-EDTA, but it might be more complexe than that and I would be interested ot know more !
Bénédicte Giroudot Thanks for your comment! We eventually determined that cells were contaminated by mycoplasma via PCR test, and thus resulted in the weird cell shapes. We believed that the mycoplasma came from pancreatin, which is the only reagent that we shared among whole laboratory.
Ah, mycoplasma ... It is the one thing I didn't think about when I answered your question, but it can indeed have an impact on the morphology ! Thank you for giving me some feedback, I will keep that in mind next time.
Mycoplasma can also come from other cells, if you manipulate more than one cell line at the same time under your BSC, and I have also heard of people who have a lot of mycoplasma on their skin and hair (quite normal I think), so much that when they prepare and run a PCR for mycoplasma detection, all the samples including the negative control turn out positive ... I guess, in this situation it may be really easy to contaminate your cultures !
I hope you will find a way to get rid of them !
Good luck with your experiments and your culture.
Bénédicte Giroudot Thanks for your suggetions! We further tested a series of samples, including different cell lines, cells cultured with different medium and culture dishes in our laboratory. It looks like all reagents were fine, but cells that cultured in 9cm*9cm culture dish turned positive in PCR
test. We believe that the culture dishes were contaminated due to collegues' improper operation, cause they didn't sealed the packing bag in time after using it.
Since then,I started to add anti-mycoplasma agents to culture medium and my SY5Y cell line is slowly growing in regular shape, for now. Meanwhile, It is curious that some other cell lines like PC12(raised by collegues) were proved to be contaminated by mycoplasma as well. However, we hadn't spotted any abnormalty of PC12 until we send it to test, which means SY5Y is the only cell line that is vulnerable to mycoplasma infection.
Hello Andreas Alcatraz ,
In fact some cell lines are more sensitive to mycoplasma infection than others. For some it is visible that they have a problem (morphology, growth rate) but for some others it is not. In any case, this is why regular testing is important, as well as precautions when manipulating different cell lines, so that you don't contaminate more sensitive cells.
In any case, if your cells were sensitive to mycoplasma, maybe they will also be sensitive to the treatments : some experimental results are affected by these anti-myco treatments, especially if the cells are difficult to treat (= more resistant mycoplasma).
Good luck with everything, because mycoplasma are not an easy problem when you are working with cells !
- Badges
- Science method