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HEKs suitable for FACS?


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#1 Taranaki

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Posted 01 March 2011 - 12:33 AM

I want to analyse the effects of siRNA-mediated Knockdown of my protein on cell cycle. Are HEKS suitable for cell cycle analysis? I just now that a similar experiment has been done in my lab with MEFs (mouse embryonic fibroblasts) and those were absolutely not suitable for flow cytometry.

Thanks!

#2 Rsm

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Posted 01 March 2011 - 11:36 PM

What do you mean with "not suitable"? I have been using MEF and HEK for FACS since 7 years now, and never had any issues with them not being suitable. Not like ES cells, for example. HEKs are even better than MEFs, because they don't attach that well, so you can just shake them loose (I would recommend 3mM EDTA in PBS, works very well). Also, transfection eficiency is much higher.
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#3 Taranaki

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Posted 02 March 2011 - 08:08 AM

Another student used some MEFs for FACS and got completely different results from time to time, because the MEFs he used had different origins and behaved differently in regard to proliferation etc. Are your results always consistent with MEFs?

But good to know that HEKs are suitable.

Thank you!

#4 Rsm

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Posted 03 March 2011 - 12:03 AM

Another student used some MEFs for FACS and got completely different results from time to time, because the MEFs he used had different origins and behaved differently in regard to proliferation etc. Are your results always consistent with MEFs?

But good to know that HEKs are suitable.

Thank you!


That the depends on what MEFs you are using. If you have real homemade MEFs, then you shouldn't use them after passage 6 or 8, they start to senesce. If you use 3T3 (immortalized MEFs), you will have much more reproducible data. HEKs are quite fine with FACS, but be aware that they are human origin, so your siRNA sequence for example is different from MEFs.
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