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





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?





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