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

 

Mara Rocchi BVM&S, PhD

Flow Cytometry Manager

Moredun Research Institute

Pentlands Science Park

Bush Loan, Penicuik

EH260PZ

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

www.bloodsystems.org

www.bsrisf.org

 

________________________________

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?

 

Thanks,

 

Sheila


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