I am trying to grow mESCs in defined contitions using N2B27 + 2i + LIF. I was growing the cells in these conditions in another lab and it worked perfectly well, but when I try it in my lab cells don't attach too well and colonies detach from the plate.
I've tried coating with gelatin for longer periods and increasing the amount of BSA in the medium, but non of these worked.
I attach the media formulation that I use.
Thanks!
Ariel
Dear Ariel:
Defined media are never as good as they claim, but they should attach.Check teh glucose concentration you are using.
During a period some years ago I did the opposite: form aggegates using bacteriological dishes, under these conditions we could form aggregates and control the size of teh aggegate by changing glucose concentrations.
Hi Ariel
I am not an expert since I am new in the field, but we have also noticed that before for some cells, and asked the question to some people in the Stem Cell Institute in Cambridge. Apparently some strains do that. Which strain are you using? Also we have that some cells that do and some cells that don't, so it might not be a matter of medium/gelatin/dish. Also, it depends if the sticking to the bottom is actually important for your experiments or not (for live cell imaging it's important but not for qPCR for ex), but my labmate has cells that float but are still growing very nicely and forming nice round colonies. So it doesn't mean that the cells are unhealthy.
Cheers, and good luck
Ag
Hi Ariel
Yeah, I guess right now what you can do is to do some QC,
1. Check with the tissue culture grade petri plates, get samples from a good vendor or so.
2. Check with the gelatin you are using, and also the concentration. May be change the concentration from 0.1% to 0.5%.
Best
Abhishek
Are you washing your cells with PBS containing Ca and Mg or without them? No Ca and Mg in PBS causes cells to detach.
Hi all, thanks for your answers!
Bernat, I've checked the glucose, particularly in the DMEM:F12 and it is high glucose, as it is supposed to be.
It might be that here I am using a different (and cheaper) brand of plates. I've tried both with Nunc and with Biofil. At first they attach, but eventually they form big colony balls that detach. I agree with Agathe that even though they are in suspension, they are in "good health"; I've even differentiated them to neural precursors and in that protocol they attach well. I am using a W4 mES cell line.
I usually wash the plates with PBS w/o Ca and Mg, but prior to tripsinization, so I want them to detach in that situation. I don't wash them regularly when they are growing.
I am going to try with different plates and as Pavan suggested increasing the concentration of gelatin to 0.5%.
Thank you all so much! I'll keep you posted.
Ariel
Hey again
Yeah, plate brand can be tricky. We found here that Nunc clearly does not perform as well as Falcon, by far. So we buy everything Falcon and it improves a lot of things (for example when you thaw cells you have way better survival in Falcon plates or 6-wells than Nunc...
Hi Agathe,
Actually, I used to use Falcon multiwell plates in the previous lab, when cells attached. So I am definetely going to try with that brand.
Thanks a lot!
Ari
I have tested the same mESC cell line in falcon and greiner bio one dishes. the cells were detaching in greiner dishes but were most of the time fine in falcon dishes.
Bests,
Arefe
Yes, I see the same thing. Now I routinely use Corning plates and they work fine (Falcon is now a corning brand i think).
Thanks and good luck!
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