4 replies to this topic

[themoon]

Hi,

This is probably a stupid question... I have recently purchases a U87 cell line and I'm noticing some white spots in my cultures when viewed with the naked eye - I have not previously seen this characteristic when I had cultures of the same cell line a while ago. I think it's probably normal, as when viewed under the micro, it seems as though it's the natural way these cells grow. I have attached a pic. They also appear to have a slightly lower adherence compared to before. Could this be due to trypsining for too long? If so, what can I do to restore them?

Thanks,

any help would be greatly appreciated.

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[gfischer]

It looks like the cells are clumping together, which is apparently not uncommon for this cell line.  A quick Google search yielded this link that may be helpful:

Solutions to U87 Cell Clumping

[themoon]

gfischer, on Jul 6 2009, 06:31 AM, said:

It looks like the cells are clumping together, which is apparently not uncommon for this cell line.  A quick Google search yielded this link that may be helpful:

Solutions to U87 Cell Clumping

Hmm, I've checked all those parameters the document suggests to check and I still get the same thing. But thank you very much for your help! Now I know it's a common problem and not some anomaly At last I have some piece of mind. I'll keep an eye on them for the next week.. hopefully they go away

thanks again!

[rhombus]

themoon, on Jul 6 2009, 03:08 AM, said:

Hi,

This is probably a stupid question... I have recently purchases a U87 cell line and I'm noticing some white spots in my cultures when viewed with the naked eye - I have not previously seen this characteristic when I had cultures of the same cell line a while ago. I think it's probably normal, as when viewed under the micro, it seems as though it's the natural way these cells grow. I have attached a pic. They also appear to have a slightly lower adherence compared to before. Could this be due to trypsining for too long? If so, what can I do to restore them?

Thanks,

any help would be greatly appreciated.

I totally agree with gfischer, the cells are clumped together and it is NOT contamination
Generally when you split/passage cells, getting single cell suspensions is important i.e. if 30% of the cells are in large clumps, the cells in the middle of those clumps will die, therefore reducing the total number of viable cells AND increasing population doubling times. Cells are fairly robust, so:

Increase the trypsinisation time
Increase the trituration of the suspension
Always passage before the cells are fully confluent...this also reduces the chances of clumping.

Hope this is useful

Uncle Rhombus

[themoon]

rhombus, on Jul 7 2009, 03:09 AM, said:

themoon, on Jul 6 2009, 03:08 AM, said:

Hi,

This is probably a stupid question... I have recently purchases a U87 cell line and I'm noticing some white spots in my cultures when viewed with the naked eye - I have not previously seen this characteristic when I had cultures of the same cell line a while ago. I think it's probably normal, as when viewed under the micro, it seems as though it's the natural way these cells grow. I have attached a pic. They also appear to have a slightly lower adherence compared to before. Could this be due to trypsining for too long? If so, what can I do to restore them?

Thanks,

any help would be greatly appreciated.

I totally agree with gfischer, the cells are clumped together and it is NOT contamination
Generally when you split/passage cells, getting single cell suspensions is important i.e. if 30% of the cells are in large clumps, the cells in the middle of those clumps will die, therefore reducing the total number of viable cells AND increasing population doubling times. Cells are fairly robust, so:

Increase the trypsinisation time
Increase the trituration of the suspension
Always passage before the cells are fully confluent...this also reduces the chances of clumping.

Hope this is useful

Uncle Rhombus

Thanks so much for the handy tips!! I've read a few sources and some say that trypsinising for too long may also be a cause.. and I think this may have occured in my case.

Thanks again!