FW: PBMC from Rabbit Spleen

Hirschkorn, Dale dhirschkorn at bloodsystems.org
Thu Nov 6 13:30:49 EST 2008


Hello Everyone,

 

Is there any one out there in the flow community that has experience
with Rabbits and in particular their spleens?  I have co-worker that is
having a problem separating cells-see her procedure and questions below.

 

Thanks in advance for any and all advice!

 

Dale Hirschkorn, MT(ASCP)

Blood Systems Research Institute

270 Masonic Ave

San Francisco, CA  94118

(415) 749-6672

www.bloodsystems.org

www.bsrisf.org

 

________________________________

From: Keating, Sheila 
Sent: Thursday, November 06, 2008 10:15 AM
To: cyto-inbox
Subject: PBMC from Rabbit Spleen

 

 

 

Hi Dale,

 

It would be great if you could send this problem out to your experts to
see if anyone has any suggestions.

I had a terrible time getting PBMCs from rabbit spleens.  Here's my
procedure and where it all went wrong:

 

I received rabbit spleens sent overnight in PBS, kept on cool packs.

I cut them into small pieces, and using the back of a syringe plunger,
pushed the tissue into a 110uM cell sieve to release the cells.  The
sieve was in a petri dish with PBS, I could see there were RBCs and
other cells moving into the the PBS.  Next I layered this suspension
onto Ficoll.  I used Histopaque 1083.  I spun for 400Xg for 30 minutes
and when I pulled the tubes out, all the suspensions were still sitting
on the ficoll.	I spun again, 500Xg for 30 minutes and still nothing.  I
tried aspirating the mass that was sitting on the ficoll and it had
turned into a gelatinous gloop.  It looked like there were white cells,
but they were embedded in the gel and could not be separated. What did I
do wrong?  How could I fix it? I was thinking, shipping spleens at room
temperature, adding DNase or collagenase? That's all I can come up with.
I would appreciate any suggestions since I am stumped.	I got most of my
advice from mouse spleen researchers and none of their suggestions
seemed to work. Has anyone worked with rabbit spleens before or rabbit
PBMCs in general?

 

Thanks,

 

Sheila

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