FW: PBMC from Rabbit Spleen
dhirschkorn at bloodsystems.org
Thu Nov 6 13:30:49 EST 2008
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
From: Keating, Sheila
Sent: Thursday, November 06, 2008 10:15 AM
Subject: PBMC from Rabbit Spleen
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?
-------------- next part --------------
HTML attachment scrubbed and removed
More information about the Cytometry