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Two distinct populations observed on a homogenous cells

flow cytometry homogenous populations

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#1 Thomson

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Posted 28 November 2018 - 04:02 AM

Hi all, I often get two populations from my results of electroporated cells compared to non-electroporated control (pic attached). Left is non-electroporated,, right is electroporated.

 

Electroporation conditions:

3 million cells, 5ug plasmid, exponential (250V, 950 uF, Ω infinite , cuvette 4mm ).

 

What could be the reason?

 

I don't think it's doublet as the fluoresence intensity of both population almost similar when measure individually. 

 

Thanks.

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Edited by Thomson, 28 November 2018 - 04:23 AM.


#2 OldCloner

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Posted 28 November 2018 - 08:50 AM

Hi Thomson,

 

First, I have never run flow cytometry myself, but while you are waiting for a real expert, I would postulate that your post-electroporation cells are a mixture of cells with the plasmid, and cells that did not receive the plasmid. That would be 2 populations. I don't think you get 100% conversion on electroporation, do you? (My experience is more with bacteria than cultured cells.)

 

As a control you could always try a dummy electroporation, with no plasmid added.

 

good luck!







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