PBMC from Rabbit Spleen

Mara Rocchi Mara.Rocchi at moredun.ac.uk
Wed Nov 12 03:19:33 EST 2008

Dear Dale, 

Please find attached a protocol that one of my colleagues uses for the isolation of rabbit PBMCs from blood. For spleens, I would dilute the cells ½ in Ca++/Mg++ free PBS before the buffy coat step.

The main difference with your protocol is the use of a Ficoll of a different density and the centrifugation speed.

Best regards



Mara Rocchi BVM&S, PhD

Flow Cytometry Manager

Moredun Research Institute

Pentlands Science Park

Bush Loan, Penicuik


Scotland, UK



Think before you print. Save paper and help the environment



From: Hirschkorn, Dale [mailto:dhirschkorn at bloodsystems.org] 
Sent: 06 November 2008 18:31
To: Cytometry Mailing List
Subject: FW: PBMC from Rabbit Spleen


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: Hirschkorn, Dale
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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