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Vials Liquid problem with thawing cells from liquid nitrogen? I took out the vial of MCF7 cells from liquid nitrogen and then left it at room temperature for 30 min to thaw, then I added media+10...


Vials Liquid

Vials Liquid
problem with thawing cells from liquid nitrogen?

I took out the vial of MCF7 cells from liquid nitrogen and then left it at room temperature for 30 min to thaw, then I added media+10% FBS and centrifuged to remove DMSO and then resuspended the pellet in fresh media and then cultured it....now it is almost 24 hours but the cells are not attaching. what is the problem?

I agree with Vishal and Yada.

The problem could be in the freezing. In my lab, we use a controlled rate freezing device (we call it a Mr Frosty, for obvious reasons). It's pretty much a wide, shallow Nalgene bottle filled with isopropanol, with a cryotube rack immersed inside. You put the tubes in the rack and place it at -80. The alcohol insulates the tubes, to an extent, and slows the freezing process down to a more healthy level.

It's more likely that the problem lies with the thaw, though. You want to minimize the amount of time that cells are metabolically active in the DMSO. When you thaw, you should place it in a 37 degree water bath (you should hold onto the cryotube so the cap doesn't immerse and potentially contaminate the cells). As soon as the last little bit of ice disappears, you should add the media.

I've found that the cells usually attach better if you don't spin them out ahead of time, since immediately after thawing, your cells will still be pretty fragile. This depends on the amount of media that you're plating in, though. If you're plating 1ml of cells into 15-20ml of media, you should be fine. Just make sure to change the media at the end of the day or on the following morning (depending on how fast your cell line attaches). If this isn't feasible, though, just make sure that you spin them as gently as possible.

 
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