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CFSE assay data analysis


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#1 Funny

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Posted 14 September 2011 - 07:48 PM

I have done CFSE assay and have data, dont know how to analyze. Anyone with any experience on this?
I have the dot blot, need to analyze the proliferation

Edited by Funny, 15 September 2011 - 03:47 AM.


#2 Rsm

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Posted 15 September 2011 - 01:30 AM

What do you want to find out? Proliferation history, cell tracing in vivo, metabolism?
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#3 Funny

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Posted 15 September 2011 - 03:48 AM

I have the dot blot. Need to know the proliferation rate

#4 Rsm

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Posted 19 September 2011 - 07:02 AM

It is claimed that you'll get distinct peaks, each of around half the intensity of the one before. Then you should have one control, which is "no proliferation" and sets your t:0. This should overlap with some of your cells, showing non-divided cells. The next peak to the left is then 1 division, then the next peak 2 divisions etc.

However, I have never observed something like that. It was always a big bobbly mess. Also the intensity drops dramatically within the first 4h due to metabolism of CFSE. Furthermore, signal depends extremely on cell size, more than on proliferation.

Taken together, counting cell divisions with CFSE works if you have extremely homogenous cells, same size, same cell cycle stage. Like quiescent T cells. But it will never work (at least for me) with cultured celllines or normal primary cells...

Maybe you can show us your dot blot? That may be easier...
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