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Help!my 293T stop growth and become slim 24h after 1:3 splitting - unless you change new medium for them 24h after splitting (Sep/09/2010 )

The 293T that I cultured are very likely to stop growth and become slim.To be specific,after being splitted 1:3 to 10cm dishes with 7ml of medium per dish,they won't become confluence after 48h or any longer,unless you change new medium for them after 24h.Actually,they will stop growth about 24h after seeding and will become slim to form some kind of web-like pattern in the dish with small areas unoccupied. Moreover,the surface of cell is kind of dirty/dusty,with many black spots and small bubbles on them.Meanwhile,the medium is likely to turn yellow.
Do you have any idea as what may be the problem?Other than too many passages...

-AllenChiu-

Sounds very much like contamination. Does your media also go cloudy? Can you post a picture?

-leelee-

leelee on Fri Sep 10 05:55:19 2010 said:


Sounds very much like contamination. Does your media also go cloudy? Can you post a picture?

I don't think there are any contamination as there are no living things except 293T..

-AllenChiu-

AllenChiu on Fri Sep 10 05:51:36 2010 said:


The 293T that I cultured are very likely to stop growth and become slim.To be specific,after being splitted 1:3 to 10cm dishes with 7ml of medium per dish,they won't become confluence after 48h or any longer,unless you change new medium for them after 24h.Actually,they will stop growth about 24h after seeding and will become slim to form some kind of web-like pattern in the dish with small areas unoccupied. Moreover,the surface of cell is kind of dirty/dusty,with many black spots and small bubbles on them.Meanwhile,the medium is likely to turn yellow.
Do you have any idea as what may be the problem?Other than too many passages...

Hi AllenChiu sounds like a problem with the medium you are using, it might not be providing enough nutrients for the cells you are using. The change in color indicates that the glucose is being consumed at a high rate by the cells dividing resulting in a decrease in pH and therefore the change in color. I would also suggest using a growth supplement adding about 10% supplementation. The growth supplement should provide the extra growth factors and energy source your cells require you can contact Invitrogen or try Genial Genetic Solution (I have used their BMGS growth factor to help with our bone marrow cell lines)...they are very good. Hope all goes well.
regards

-stuart-