DRAQ5 diffusion

[Louis Villeneuve]

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)

[hidden email]

[Sylvie Le Guyader-2]

Hi Louis

 

I would try as described labelling the aorta cells, washing them for 2 rounds, resuspending them as for injection then spinning them down again and injecting only the supernatant. If you still get endothelial and muscle cells weakly labelled, it means you don’t wash enough (the dye might somehow get concentrated in those cells) otherwise it means that your label cells label the others.

 

Med vänlig hälsning / Best regards

 

Sylvie

 

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Sylvie Le Guyader

Dept of Biosciences and Nutrition

Karolinska Institutet

Novum

14157 Huddinge

Sweden

[phone]

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: 18 November 2008 16:47
To: [hidden email]
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)

[hidden email]