DNA stain for viable FACS cell sorting? - (Sep/16/2005 )

Hi,

I was wondering if anyone knows if there is a DNA stain that can be used to stain the DNA content of cells for FACS cell cycle analysis and permits the recovery of live sorted cells.

Seems from my research that one of the Hoechst dyes might be my best option but I'm open for any suggestions.

Thanks for any help you can provide

LeserattePD

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Hi,

With no more information about the cell sorter that you could use and the LASER it has I recommend you to try Molecular Probes Vybrant Dyes. There are different Vybrant dyes that emitts and absoves at different wavelenghts and they need no permeabilization or fixations, just add dye and incubate for a shirt time and read in a flow cytometer. I use Vybrant violet with good results in a Violet LASER but you could use 488 LASER with other Vybrant Dyes.

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[/quote]
Hi,

With no more information about the cell sorter that you could use and the LASER it has I recommend you to try Molecular Probes Vybrant Dyes. There are different Vybrant dyes that emitts and absoves at different wavelenghts and they need no permeabilization or fixations, just add dye and incubate for a shirt time and read in a flow cytometer. I use Vybrant violet with good results in a Violet LASER but you could use 488 LASER with other Vybrant Dyes.
[/quote]

Thanks a lot for your help, but it seems that these dyes are lipophilic stains ie they stain membranes - I need something that stains DNA because I need to assess DNA content and select cells dependent on what cell cycle phase they are in.
FACS is not a problem - I can get access to different ones with different lasers.

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[quote name='LeserattePD' date='Nov 10 2005, 02:13 PM' post='30623']
[/quote]
Hi,

With no more information about the cell sorter that you could use and the LASER it has I recommend you to try Molecular Probes Vybrant Dyes. There are different Vybrant dyes that emitts and absoves at different wavelenghts and they need no permeabilization or fixations, just add dye and incubate for a shirt time and read in a flow cytometer. I use Vybrant violet with good results in a Violet LASER but you could use 488 LASER with other Vybrant Dyes.
[/quote]

Thanks a lot for your help, but it seems that these dyes are lipophilic stains ie they stain membranes - I need something that stains DNA because I need to assess DNA content and select cells dependent on what cell cycle phase they are in.
FACS is not a problem - I can get access to different ones with different lasers.
[/quote]

These are dyes that binds speciphically DNA. They goes trought membrane to nucleus and stain DNA double strant only. You don't need RNAsa nor fixation. Look the atachment file of the product.

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you could try using Hoechst 33258

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a simple propidium iodide labelling is sufficient
select cells that have exclude the dye!

Seb_

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I'm not quite following the argumentation of how I would know the cell cycle phase from the fact that PI is excluded from the cells? I always thought PI stained dead cells only...

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Thanks a lot. I must have gotten the wrong product when I checked the molecular probes catalogue. Sounds like these dyes are really good. Do you have any experience with how toxic these stains are to cells? That little tidbit of data isn't mentioned in the sheet...

So far I've had good results (ie cells surviving relatively well) with using 5ug Hoechst 33342 per ml and 1 million cells. Staining was good enough for a sorting. Cells have a lag phase of around 2 days but will start growing again after that.

Now I just have to find a non-toxic method of cell synchronization.

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