I am working with the R1-ESC line. I was growing them on feeders on mitomycin C inactivated MEFs using FBS from Invitrogen (South American Origin). They were growing fine in this FBS. Recently, upon revival of these cells in medium cpomrised of better quality FBS (certified, US origin), the cells appear apoptotic and don't proliferate. Instead of forming smooth colonies, they colonies appear whitish all over with unsmooth borders. I follow all other practices like feeding the medium daily and passaging every 2days. Could someone please help me out with this? I tried using ES-qualified FBS too (Invitrogen) but even that led the cells to show an appearance as mentioned above. I supplement the ESC medium with 15%FBS, NEAA and beta-mercaptoethanol.
Have you tried another batch of FBS? Test it first, a great chance it contains impurities.
The cells are really picky, we usually test different FBS from different suppliers before changing definitely. During the test, the morphology and proliferation of the cells were really different even with the FBS supposed to be specialized for the ESC and, endotoxin free. Moreover the cells need some time to adapt to their new environment, just like any other organism. Since when do you grow them in this FBS? I would suggest you to test different FBS before changing it.
Dear Fleur,
I have been growing them in the US origin FBS for quite some time. In the middle, due to non-availaibility of this FBS, I had to grow them in the South American origin one and the cells adapted to them very easily. Now, again when I want to grow them in the US origin FBS, they are no longer growing properly. As you said, even in the ESC qualified FBS, the colonies are appearing whitish.
Check your cells with another batch of FBS, but also test them for mycoplasma. Keep a test well with the same media for 2 days and on the third day check for mycoplasma.
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