Re: DRAQ5 diffusion (Tech. Support response)

[Roy Edward]

Dear Louis

 

Sorry, I only just saw your question to the list.

 

I am not suprised that you have this secondary staining.  This is most probably a function of the fixation process. 

This would mobilise some dye that can potentially then get into the surrounding unstained cells, hence the differential staining intensities which might be a useful marker anyway! 

Live-cell permeant dyes (like drugs) are typically lipophilic and there may be the release of a small reservoir of dye associated with lipid rich membrane-like structures during the fixation process.     

Also, the natural equilibrium of the dye will be disturbed upon fixation of the chromatin so that the tiny mobile dye fraction proximal to the DNA cannot interact with the DNA so well and then the exchange to unoccupied sites in other cells could occur.

  

If you can by any means avoid fixation this might resolve the issue.

 

Another means of fixation might improve the situation also.  Examples of different fixatives used with DRAQ5 can be found at:

http://www.biostatus.com/application/confocal_microscopy/fixed_cells__nuclei/

 

Titration of DRAQ5 downwards might reduce the effect since the non-bound dye fraction will be so much lower and in all likelihood this "donor" effect would be ratiometrically reduced. I would try to limit the cell washing to a minimum after staining as this would be a significant variable in the final staining intensity of the target cells.

 

Any live cell "donor" transfer effect should be seen in a control experiment - e.g. a mixture of live stained and unstained cells using similar time parameters/conditions to those you have established.  I would be surprised if you see that since, unlike some other live-cell permeant nuclear counterstains, DRAQ5 is demonstrably temporally very stable in its DNA binding (i.e. it is not seen by the ABCG2 pumps).  This has been shown for flow cytometric DNA cell cycle analysis and for microscopy.  I can provide references, etc. on this if you need it. 

 

Please don't hesitate to contact our tech support directly for assistance in future ([hidden email]).  We will always answer questions within 24 hours.

 

Best regards

Roy

 

Roy Edward

Biostatus Ltd

OFFICE: [phone]; [hidden email]; www.biostatus.com

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From: [hidden email] [mailto:[hidden email]]
Sent: Tue 18/11/2008 15:46
Subject: DRAQ5 diffusion

Hi everyone,

We are using DRAQ5 to label cells that will be injected inside a mouse aorta.  The aorta has been previoulsy extract from the animal and the incubation of the labeled cells inside the aorta is short  , about 5 min. Then the whole aorta is fix with 4% paraformaldehyde.

 Cells (mesenchymal cells from rats)  are labeled with 2uM DRAQ5 (for 1 million cells) and incubation is carried out at 37°C for 3min.  2 round of centrifugation (5min, 1200 rpm,  using 5 ml of media) are done for washing.  

We see  endothelial cells  and in a weaker manner, the smooth muscle cells labeled with DRAQ5 despite we were expecting only the injected cells to be labeled.  The short incubation time of these cells inside the aorta tend to tell us that cells cannot migrate trough the media so fast!!!!!

Finally my question, can DRAQ5 diffuse from a previoulsy labeled cells and label surrounding cells?  

Thanks for your help!

Louis

Louis Villeneuve
Research Associate- Confocal Microscopy
Heart Montreal Institute- Research Center
5000 East Belanger
Montreal (Qc), Canada
H1T 1C8

514-376-3330 ext 3511
514-376-1355 (Fax)

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