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Why GFP/YFP+ neurons couldn't be seen at cytometer after mouse brain dissoci

Percoll YFP Papain Flow cytometry Neurons

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#1 Marinavv

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Posted 19 June 2015 - 03:15 AM

Hi everyone! I'm trying to isolate parvalbumin-positive neurons of hippocampus and cortex in transgenic mice with YFP expressed only in parvalbumin neurons (Cre/Loxp method). I want to extract RNA from these cells in order to make transcriptomics.
My protocol is based on decapited mice, extract brain, disintegratation with papain solution, 70 micrometer filtration, a percoll gradient to eliminate debris (I tried a lot of percentages: 30%, 40%, 90%..) and finally I directly go to FACS with all brain cells washed after percoll to eliminate any residue. But I had never seen any YFP-positive cells: control mouse (YFP-) and mutant mouse (YFP+) show the same pattern, no fluorescence.

I also tried to use other mice expressing GFP under another promoters (GAD1-EGFP,...) but it was the same: no fluorescent cells found.

Does anybody knows why GFP/YFP could be lost in my preparation? Because I see papers where they use this type of transgenic mice and have no problems with seeing the GFP at cytometer and they don't do anything different from my protocol. HELP please! I've been trying different protocols a long time and nothing changes.



#2 bob1

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Posted 20 June 2015 - 01:28 AM

Are you fixing the cells? this can cause loss of GFP and GFP derived (e.g YFP) signal. I think it depends on the fixative though.



#3 Marinavv

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Posted 22 June 2015 - 02:17 AM

Thank you for you answer, but I didn't use any fixative because I know their problem in seeing GFP, so I go directly to flow cytometry without doing anything apart from disintegration and percoll. It has to be another thing..



#4 bob1

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Posted 22 June 2015 - 01:02 PM

Do you have a GFP positive cell line (transfected?) that you could put through the same procedure (probably without papain digestion)? It sounds like you are either losing the signal somewhere during processing or the FACS isn't working as you expected.

 

Could you take some of the cells and put them under a fluorescent microscope?







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