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Advice Needed about Confluency


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#1 Apop Culture

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Posted 22 October 2013 - 07:32 AM

Hey all,

I've just started doing culturing of HEK293T and Cos7 cells, but I don't know what the different stages of confluent look like. I understand the concept, but my visual mind can't comprehend without a picture. Therefore, I was hoping that someone might know of a good website or have some pictures of cells that are 30%, 50%, 70%, 80%, 100%, and over- confluent. Just so I can get an idea of what each step looks like. Thanks for reading and helping!

 

 



#2 bob1

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Posted 22 October 2013 - 12:37 PM

Well, 100% should be easy - this is where the dish is completely covered with cells (for most cell types at least).  Over confluent is where the cells start growing over each other.  50% is halfway between no coverage and 100% (obviously).  The best way is to get someone experienced to look at your cultures and get them to estimate confluency, then have a look yourself.  Note that this process is highly variable, if you got 10 different people to look at them, you would probably get 10 different answers.

 

try: here



#3 wpeng

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Posted 28 October 2013 - 08:17 AM

Hi, I'm completely new to cell culture.  My mentor told me once that 90% to overconfluent cells develop mutations and are no longer reliable for experiments.  I have read a number of cell culture manuals, and the main problem with overconfluency is that cells are no longer in the log phase of the growth curve and may take a long time to recover growth after passage.  I have been unable to find any information about mutations or reliability with experiments.  What is the truth of the matter?

 

Thanks!



#4 bob1

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Posted 28 October 2013 - 11:48 AM

@wpeng - it is all cell line dependent, some cells will not survive after reaching confluence, others will happily grow in an over-confluent state (e.g. HeLa), while others will stop growing once confluent, but then be able to grow again when split.

 

The reliability for experiments is a difficult one, it all depends on what you are looking at, but for most purposes, actively growing cells that have been treated in the best manner possible (i.e. split when needed, fed when needed, incubated under good conditions etc.), will give you reliable and most importantly reproducible results.  Cells that have reached confluence may not do this, due to variable growth rates and metabolic activities.

 

I don't know about mutations so much associated with confluence, but there are changes in cell behaviour with the age of the culture, which I think is associated with methylation patterns of the DNA and probably mutation too (it would pay to check me on that though!).  For this reason you shouldn't keep cells up for more than 10-15 passages and try to make new stocks at as early passage as possible.



#5 wpeng

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Posted 28 October 2013 - 01:22 PM

Thank you very much.  I was asking because I had a malignant cell line grow faster than I expected over the weekend, and I did not want to discard it because we do not have a lot of that cell line in storage.  I guess it would still be safer to discard it and start anew.






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