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H9c2 cell contamination?


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#1 szavitsa

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Posted 29 June 2009 - 09:04 AM

I have a problem with my H9c2 cell culture, I hope somebody could help me with this,I would be most grateful.
I see big, white, circular shapes at the sides of my flask (not moving) and in my cell culture (floating, I'm not so sure). Is it a contamination, like yeast for example?
I have to admit that I sometimes keep my culture confluent and when I trypsinize my H9c2 cells, I see aggregates.
I hope somebody could give me an answer, if anyone has came across with such a case.I would truly appreciate it.

#2 bob1

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Posted 29 June 2009 - 04:19 PM

How large? If you look at it closely (microscope if necessary) is it fuzzy/hairy? If so, it is a fungal contamination - throw out your cells and start again.

It could be cell aggregates, but you should be able to see that it is cells attached to one another if this is the case. If so, you need to subculture your cells more frequently, as you are putting selective pressure on the cells to be able to grow in overcrowded conditions.

A picture would be helpful!

#3 szavitsa

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Posted 29 June 2009 - 10:47 PM

How large? If you look at it closely (microscope if necessary) is it fuzzy/hairy? If so, it is a fungal contamination - throw out your cells and start again.

It could be cell aggregates, but you should be able to see that it is cells attached to one another if this is the case. If so, you need to subculture your cells more frequently, as you are putting selective pressure on the cells to be able to grow in overcrowded conditions.

A picture would be helpful!


It's not fuzzy neither hairy. It is approximately 100-500 times bigger than the size of a single cell and if I look it in the microscope it seems more like a compact mass, cells attached to each other (the outline has the shape of round cells stuck together). Unfortunately, I'm not able to give you a picture as we don't have camera.
I study signaling pathways. Do you believe the way I split cells affects my experiment results in any way?
Should I throw out this culture anyway?
Thank you so much for your advice!!!!

#4 bob1

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Posted 01 July 2009 - 04:25 PM

Everything you do to the cells affects them in some way! It is best to routinely culture your cells so that they do not become too densely packed as this will select for cells that can grow well under these conditions. It will also (and it is a big one) ensure that your results are more repeatable - if you try and experiment with overgrown cells and get one result, and then try it with less densely packed cells, you may well get a different result. Keeping them at a narrow range of densities (30-70% say) will ensure that you don't have these sorts of problems.

Aside from the experimental, it will also help you detect contamination, see the general health of your cells, and ensure that they are fed properly.

#5 szavitsa

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Posted 02 July 2009 - 02:01 AM

Everything you do to the cells affects them in some way! It is best to routinely culture your cells so that they do not become too densely packed as this will select for cells that can grow well under these conditions. It will also (and it is a big one) ensure that your results are more repeatable - if you try and experiment with overgrown cells and get one result, and then try it with less densely packed cells, you may well get a different result. Keeping them at a narrow range of densities (30-70% say) will ensure that you don't have these sorts of problems.

Aside from the experimental, it will also help you detect contamination, see the general health of your cells, and ensure that they are fed properly.


Thank you so much for your helpful advice! You see, before keeping my culture overcrowded, I was subculturing my cells in such a density that they didn't proliferate well (below 40%) so that they couldn't detach from the bottom of my flask when I trypsinized them.
I will try to keep them in an appropriate density as you said. I'm so grateful for your advice!!!!! :-)




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