7 replies to this topic

[nly87]

Hola!

A friend has cultured MCF-7 cells from a frozen vial and had used RPMI + 10% FBS + 2mM L-glutamate. The following day we realized that majority, if not all, are dead (but still adhering). I think it's because of her media which is about 6 mths old. So we have changed the media to DMEM(with L-glutamate) + 10% FBS + 1% Penicilin + 1X sodium pyruvate. But we see no change still. Maybe some hopeful clumps of cells. Should we wait for another day or just curse at it?

Lee

[bob1]

How do you know they are dead?

If they are particularly unhappy now would you still want to use them after such a drastic selection/stress event?

[nly87]

They are all circular or irregular. Doesn't reflect the normal epithelial-like under the microscope. I was just hoping that some will still live and start to proliferate..

[bob1]

It is highly unlikely that they are dead if they are still attached.  Dead cells typically detach and float.

It is certainly unusual for MCF-7 to not attach well.

In this sort of situation you should always ask yourself:

"How will {whatever is happening to the cells] affect my experiments?  Would I still be comfortable using them knowing that the event might influence my results?"

[GNANA]

As Bob 1 said, it is not advisable to use those cells for any of ur experiments even if you recover them from the present condition.  Just throw them of and start from a new vial.

[nly87]

Ok thanks! Btw, I was just wondering if floating cells = dead cells and if dead cells = float.. Or is it just for some cell lines? Because I'm very new to culturing cells and I hear people telling me different things.

[Kamran]

I's also having similar problems with PTN-1B cells but I discarded them without any second thought.

[bob1]

nly87, on 09 September 2011 - 12:56 AM, said:

Ok thanks! Btw, I was just wondering if floating cells = dead cells and if dead cells = float.. Or is it just for some cell lines? Because I'm very new to culturing cells and I hear people telling me different things.

Neither and both - dead cells typically float, but floating cells are not necessarily dead.  There are many cell lines out there that are cultured in suspension only, and even some that have both floating and attached cells as part of the normal population (e.g. SY5Y).  The only way to determine if the cells are dead is to do a death test.  This can be as simple as a trypan blue exclusion assay, but there are many more complex and specific methods for determining how (which pathway used) the cells died.