I am now making L-cell conditional media. Our L cell growth really fast. I thaw one tube into a T75, and then split into a T175. After confluent, I split the T175 into 5 T175 flasks. Then after confluent, 100 ml media were added and let the cell sit there for 7 days. I checked the cells on the 3rd day, I found the cells is started to float, and media turns to yellow. I wonder if this means the cells do not have enough nutrition and starts to die? I am using DMEM+10FBS+Penstrep to culture them.
Looking forward to any suggestions please!
Thanks a lot!
Hello Wang Yuan
As you have mentioned the cells are being thawed into the culture flask, you may wanna check your DMSO with regards to the quality and the quantity that is been used to prepare your freezing mixture.Since your cells are growing very rapidly as mentioned it will be better if you observe them the very next day and subculture without any further delay as they die by 3 rd day. Change in colour to yellow indicates the change in pH with regards to Co2 % of your incubator. Do look into that as well
Here's a Link that may help you out :
Cheers
https://www.atcc.org/products/all/CCL-1.aspx#culturemethod
Hi Zubin,
Thank you very much for your suggestions~ :)
I am gonna to thaw another one, and do several round of sub culture before I make the media. I wonder if the cells are more stable could help them to live longer in the media.
We have other cells culture in the incubator, which are fine so far, our CO2 setting is 5%... I will keep an eye on that too!
Thank you very much~
Best
Wang
Hi Wang Yuan
If you want to make your cells more healthier and stable after thawing your frozen cells into culture flask with fresh medium leave it for a overnight incubation. Then very next day replace your medium of your T 25mL culture flask wash your cells with PBS so that there wont be any traces of DMSO left and replenish with fresh medium again.
Good Luck
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