[Cytometry] Frozen PBMCs for TBNK

Menendez, Fred A. fmenende at jhsph.edu
Wed Apr 3 07:21:40 EDT 2013


Check out references from the Multi-Center AIDS Cohort Study, the MACS. They have cryopreserved PBMC dating back to the beginning of the AIDS pandemic. They also have a very good QA/QC program  and methods for freezing and thawing PBMC. 

Fred Menendez
Flow Cytometry Core Laboratory
Johns Hopkins Bloomberg
School of Public Health
________________________________________
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] On Behalf Of Prussin, Calman (NIH/NIAID) [E] [CPRUSSIN at niaid.nih.gov]
Sent: Tuesday, April 02, 2013 10:41 PM
To: Divya Bhatia; Sundararajan Jayaraman
Cc: cytometry at lists.purdue.edu; Lukas,  Susan
Subject: Re: [Cytometry] Frozen PBMCs for TBNK

My guess is that Divya is referring to cells cryopreserved alive. Our experience is that after fixation in 4% paraformaldehyde, -80C cryopreserved cells last essentially forever (>> 5 years).

Calman
---
Calman Prussin
Laboratory of Allergic Diseases
National Institute of Allergy and Infectious Diseases/ NIH
tinyurl.com/prussin1
calman at nih.gov
________________________________________
From: Divya Bhatia [divyabhatia2 at gmail.com]
Sent: Monday, April 01, 2013 1:10 AM
To: Sundararajan Jayaraman
Cc: cytometry at lists.purdue.edu; Lukas,  Susan
Subject: Re: [Cytometry] Frozen PBMCs for TBNK

Hi,
I guess TBNK stands for T-cells, B-cells and NK cells, I did staining for B
cells (CD45+, CD19+), Tc (CD8+) Th (CD4+), and also cultured after
stimulation with PMA, Ionomycin and monensin for staining for Th1
(CD3+CD8-,IFN-gamma+), Th2 (CD3+CD8-, IL-4+,IL-13+) and for Th17
(CD3+CD8-,IL-17+,RORgt+) cells even after one month on cryopreserved cell
(stored at -80 degree). I found it would be better to stain within 15 days
after cryopreservation if you want to look for intracellular cytokines and
transcription factors. You ll get pretty good expression of surface markers
even after one month on cryopreserved cells.

Cheers
Divya


On Mon, Apr 1, 2013 at 7:07 AM, Sundararajan Jayaraman <anue2468 at uic.edu>wrote:

> It would be helpful if you explain what is TBNK. Also, how was the PBMC
> frozen, stored and how long? What is the yield of live cells after thawing
> in your hands?
>
> Thanks.
>
> --
> S. Jayaraman, Ph.D.
>
>
> On Tue, March 26, 2013 10:27 am, Lukas, Susan wrote:
> > Hi all,
> > Has anyone tried running TBNK on frozen PBMC's?  If so any pit falls?
> >
> > Susan
> >
> >
> > Susan Lukas, MPH
> > Sr. Biological Scientist
> > University of South Florida
> > Children's Research Institute
> > 601 4th Street, South CRI 4008
> > Saint Petersburg, FL  33701
> > 727.553.1259
> > 727.553.1295 (fax)
> > slukas at health.usf.edu<mailto:slukas at health.usf.edu>
> >
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>
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--
*Thanks & Regards :
Divya Bhatia
Ph.D Student*
*Department of Pediatrics
All India Institute of Medical Sciences
New Delhi, India*
*Tel. 011-26546518*
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