I thawed A20 cells and I had around 70% mortality, because there weren't freeze properly, but that's all I have to work with.
I'm cultivating them in RPMI media supplemented with 10% FBS, 1% L-glutamine, 1% pen/strep, 1% sodium/pyruvate, 0,1% b-mercapto and I added enough glucose to get a concentration of 4500 mg/L.
I've been cultivating them for 6 weeks now, and nothing happens. They grew (slowly) until I had 2 million cells in my flask and it stagnated there. When I count them with trypan blue exclusion, my cells are always alive, but I have around 2 million cells every time, no more. They don't die, but they don't grow either. I tried to transfer them in a bigger volume, then a smaller one, and that changes nothing. I also noticed they tend to precipitate and stick to the bottom of my plate/flask and they are very hard to resuspend, is that normal?
So how do I get these cells to grow? Thank you very much!
Hi! It could be few reasons of this:
1) Exhauting of culture medium (try to renew it!)
2) absence of limiting factors (for example EAA, vitamins, etc.) in the culture medium (find which one and add them);
3) too small volume for cultivation (the cells biomass must not exceed more then 20% of whole culture medium).
Hi! Thank you for your advices! Unfortunately, I did try to renew my media already and I' cultivating them in a proper volume of media. I will try to check if a limiting factor might be missing. Maybe they degraded in my media for some reason... Is FBS an important factor in this type of cell line or is glucose more important?
It's hard to say, 'cuz I haven't worked with with cell type, you must look in specialized articles.
Hi! After a couple of tries we bought a new vial from the supplier. It cost quite a bit but it worked right away!
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