Hi
Please reply me the proper solutions, I am frustrating with A549 cell lines. Whenever I have revived fresh vial from the liquid nitrogen I am not getting problem up to 3 splits after that the cells suddenly changing their shape. And they are not growing and the background is changing into black.
I have tried to change into fresh media, and fumigating every week. I am taking all aseptic precautions, but I am unable to detect the solution.
If I discus this with my PhDs and they are trying with fresh liquid nitrogen frozen one and they are watching up to 2-3 splits then they are saying they are fine. But whenever I have continue their step, suddenly their shape is changing.
Please post me proper answers.
Thanking you all........
Interesting problem! You may check if there is a mycoplasma contamination. Is the new shape is like fibroblastic appearance? If yes, they may undergo mesenchimal transition or senescence. In this case, you have to get rid of them. Also you had better to check the concentration of tyripsin. Hope it is not too much. Also check the CO2 level of the incubator. The display may sometimes show the correct percentage but it may actually not the correct level. Get it checked. Good luck!
I completely agree with Dr. Engin, it is due to microplasma contamination. See this links.
http://www.bionique.com/mycoplasma-resources/faq/avoid-mycoplasma-contamination.html.http://www.promokine.info/products/microbial-detection-elimination/mycoplasma-elimination-solutions/
I usually use A549 and I can say that they are growing really fast without any problem. In your case if you checked the medium, the percentage of Co2 of the incubator and nothing is changed it could be that you have mycoplasma contamination. I would suggest to check it. Best wishes!!
Thank you all for your suggestions. But I am using suggested DMEM high glucose media and strictly maintaining CO2. But i dont know whether it is mycoplasma contamination or not. Please suggest me complete handling procedure for A549 cell lines.
We split ours at a T/16 once per week. Check purity of cell line. It sounds maybe you have some Heps or something else in there. Mycoplasma cont can be checked with A7 plates or PCR.
I use DMEM low glucose from Lonza. I didn't have problem with this kind of medium. Maybe you could try to see if you can solve your problem
http://www.lonza.com/products-services/bio-research/cell-culture-products/classical-media/dmem/dmem-low-glucose-without-L-glutamine.aspx
madam, we are preparing the media manually. And interesting think is another cell lines like HELA, MCF-7 growing well in that. I am unable to find it.
I would suggest to prepare DMEM low glucose and try to culture A549 with it
the two posibility are
mycoplasma or cross contamination with another cell line., assumin that othre cells lines are growing in the same condition in your lab
here I am attaching the picture (photo has taken after transfer into petriplate (72 hrs later)). please find that what is that problem?
We use prepared DMEM F12 for A549 line and they growing fast as Mariangela´s. We maintain all of the mammary lines by this media.
Do you know the accumulated splits in the fresh liquid nitrogen frozen one?
It could be another reason for they change their shape
to me they don't look like A549 cells. You have a lot of dead cells. It seems that they are infected, I don't know if you have mycoplasma or biological contaminations ( yeast, mold, bacteria).
Please take a look to some info related to contaminations maybe they can help you:
http://csmedia2.corning.com/LifeSciences/Media/pdf/cccontamination.pdf
Good luck!!!
Thank you madam for your kind support. I will try to grow in L-DMEM with new vial from liquid nitrogen. Then I will notice you the status.
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