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Is it appropriate for FACS sorting


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#1 Jia

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Posted 03 October 2010 - 12:26 AM

Recently I am thinking of doing an experiment that using the Adenovirus which carry on RFP fluorescence to transfect my cells and FITC-conjugated antibody to detect the cells of my interest and then doing FACS sorting to select the RFP+FITC+ double positive cells. As you know, the adenovirus stay in the cells, whereas FITC conjugated antibody detect the membrane protein of my cells. Therefore I am not sure, if it is OK to select double positive cells in this experimental system. If it is yes, should I fix the cells before FACS sorting? Thanks so much!

#2 Rsm

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Posted 03 October 2010 - 06:37 AM

Yes, you can detect RFP and FITC simultaneously on the same cell. You should have some controls: RFP only (Adenovirus only), FITC only, unstained. I would suggest you to use ToPro3 (or DAPI if you have the laser) for life/dead cell discrimination (dead cells show autofluorescence), then you need also ToPro3 only stained cells.
It's better if you don't fix your cells. You will not be ab;e to distinguish life from dead, and maybe your surface epitope won't be recognized anymore.

rsm
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#3 Jia

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Posted 05 October 2010 - 12:03 AM

Thank you so much Rsm. I have learned a lot from your reply. I am really concern about what you have mentioned that using the antibody would have a risk of losing its epitope. The epitope I use to recognize the cells of interest is a tyrosine kinase receptor which is very important to the sorted cells. What should I do? Should I change the antibody instead?


Yes, you can detect RFP and FITC simultaneously on the same cell. You should have some controls: RFP only (Adenovirus only), FITC only, unstained. I would suggest you to use ToPro3 (or DAPI if you have the laser) for life/dead cell discrimination (dead cells show autofluorescence), then you need also ToPro3 only stained cells.
It's better if you don't fix your cells. You will not be ab;e to distinguish life from dead, and maybe your surface epitope won't be recognized anymore.

rsm



#4 Rsm

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Posted 05 October 2010 - 10:37 PM

I was talking about fixing your cells. You can use your antibody on unfixed living cells, no problem.

rsm
I got soul, but I'm not a soldier




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