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DiI staining Fixation protocol

Fluorescent dye fixations FACS

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#1 sekots

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Posted 09 March 2012 - 03:33 AM

Hi all,

I am looking to measure proliferation rate using DiI (red), a lipophilic dye. The cells I am using are A549-H2B-GFP cells that stably express GFP. I inject the A549-H2B-GFP cells with DiI and measure proliferation by FACS (DiI stain intensity will half ezch cell division cycle).

I was hoping someone has experience with fixing cells stained with DiI. I am aware that GFP can leak if fixed in 4% PFA and that it is pH dependent (7.4 is optimal?). Does DiI do the same thing? Unfortunately, as I need to travel to another site to perform FACS, my samples must be fixed and so I would like to know if anyone has experienced problems with losing DiI following PFA fixation.

Any help is greatly appreciated.

Many thanks,
sekots

#2 Rsm

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Posted 09 March 2012 - 03:39 AM

I don't think that you'll lose DiI signal after fixation, as it is a small molecular dye and covalently bound to proteins in the cell. However, I think you'll lose all of your GFP signal upon fixation (GFP as protein needs a defined tertiary structure for resonance). The best would be to use unfixed cells as a control. How long is your travel time? Cells can survive at least 2h on ice. Maybe you can handle your cells at the site of the FACS, I guess they'll have some equipment for that.
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