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Problem with growing HEK293

cell culture

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3 replies to this topic

#1 erimyes



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Posted 09 April 2017 - 08:17 AM

I have noticed my cells are growing slower than regular a week ago and I suspect about contamination.So I did staining & imaging yesterday using hoescht 33342 dye and noticed abnormal shape of nucleus in my cells. Does anyone have any idea what could be causing this? I prepared my medium on 22nd of February with following recipe. Plus I was doing transient transfection to my cells previously gfp fused plasmid that I believed to be expressing, however I also noticed yesterday that my cells without any transfection are emitting flourescence under gfp  spectrum too. I have no prior knowledge working with cell cultures so any insight would be appreciated. TIA

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#2 bob1


    Thelymitra pulchella

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Posted 10 April 2017 - 07:51 AM

The cells look fairly normal - nuclei are generally not round in adherent cells.


Growth slowing is another matter - if you can tell us what you have done with the cells and anything that might have changed (e.g. different lot of FBS recently), we will have a better idea of what might be going on. 


There is always some auto-fluorescence with cells in the green channel - and it is can be a big problem as it is almost impossible to remove. If you are looking for a faint change or minor differences in localization it might be better to go for a RFP or YFP. 

#3 Missle



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Posted 10 April 2017 - 09:43 AM

I normally don't use media for much more than a week.  If you're still using complete media from 6 weeks ago, that's not a good thing.  What seeding density are you passaging them and what is the media formulation?

#4 rkay447



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Posted 10 May 2017 - 11:24 AM

Do a mycoplasma test.  In my hands, 293T cells are very sensitive and greatly reduce proliferation (and increase adherence) with contamination.  Zooming in, I can find a few cells with cytoplasmic dots, but DAPI is the least sensitive way to detect mycoplasma. 

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