[Cytometry] Clogging from running digested mouse lungs

Victoria Best bestv at ccri.net
Tue Feb 3 17:50:30 EST 2009


Hi Ryan,

I routinely process Lung, and Skeletal muscle (for lymphocytes) using the
same procedure but clean it up with Percoll after the digestion.  Makes a
huge difference.  

-Victoria. 


On 2/3/09 1:31 PM, "Ryan Rountree" <Ryan.Rountree at bn-immunotherapeutics.com>
wrote:

> Hi All, 
> 
>  
> 
> We have been getting some clogs in our FACScalibur after running
> digested lung samples. The lungs are prepped by mincing and digesting
> with DNAse/Collagenase. Then squishing and straining through 70um nylon
> filters. There is lots of debris in the prep when you look under the
> microscope, and the pellets for 1E6 cells are much larger than 1E6
> splenocytes. But I don't think we're having any problem with
> resuspending the cells. Others on this site are using a similar
> protocol.
> 
>  
> 
> However, the day after running our samples, people have found clogs in
> the SIT. I'm going to start checking the SIT for buildup routinely now,
> but we think these samples are causing the problems. We also run a lot
> of whole blood, which tends to be viscous and could cause build-up over
> time.
> 
>  
> 
> Any advice for how to clean better??
> 
>  
> 
> This protocol is run for cleaning after our session:
> 
>  
> 
> 5 min 10% Contrad 70 on hi
> 
> 1 min 10% C-70 with the arm to the side
> 
> 1 min 10% bleach with the arm to the side
> 
> 5 min 10% bleach on hi
> 
> 1 min water with the arm to the side
> 
> 5 min water on hi
> 
>  
> 
> Leave tube of water on the SIP
> 
>  
> 
>  
> 
> Thanks for your help,
> 
> Ryan
> 
> Ryan Rountree, Ph. D.
> Research Scientist II
> BN ImmunoTherapeutics, Inc.
> 2425 Garcia Ave
> Mountain View, CA 94043
> Desk: 650-681-4673
> Fax: 650-681-4680
> 
>  
> 
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