PBMC from Rabbit Spleen
Mara.Rocchi at moredun.ac.uk
Wed Nov 12 03:19:33 EST 2008
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.
Mara Rocchi BVM&S, PhD
Flow Cytometry Manager
Moredun Research Institute
Pentlands Science Park
Bush Loan, Penicuik
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
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
To: Hirschkorn, Dale
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?
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