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strange dots in cell culture flask??


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#1 themoon

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Posted 05 July 2009 - 06:08 PM

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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#2 gfischer

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Posted 06 July 2009 - 06:31 AM

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
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#3 themoon

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Posted 06 July 2009 - 06:48 AM

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!

#4 rhombus

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Posted 07 July 2009 - 03:09 AM

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

#5 themoon

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Posted 07 July 2009 - 03:25 AM

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!

#6 science_rocks

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Posted 17 June 2013 - 04:11 AM

I know I see this topic a little bit late...but I have a qurstion too! I had the same excact picture from a mouse colon adenocarcinoma cell line, but after 5-6 passages the whole picture turned into a contaminant one! most cells were dead and the medium was blurry. do you have any idea what's wrong with my cells?

#7 bob1

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Posted 17 June 2013 - 12:36 PM

Clumps like that are usually the result of either the cells being over confluent before passage or over trypsinisation of the cells during passaging. Some cell lines grow like that as part of their normal growth conditions too. The contamination is a secondary problem (though in reality it will be a bigger problem to get rid of successfully).

Your best bet with regards to the contamination is to throw out all your medium and other cell culture reagents that you are currently using, get fresh ones out, change your lab coat, clean incubators and waterbaths. Thaw a fresh vial of cells and see how it goes.




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