[Cytometry] Frozen PBMCs for TBNK

Prussin, Calman (NIH/NIAID) [E] CPRUSSIN at niaid.nih.gov
Wed Apr 3 12:57:39 EDT 2013


Divya,

The sequence is this:

1. Isolate PBMC
2. Activate with PMA/ionomycin, BFA (or monensin) x 6h
3. Amine reactive viability dye
4. Fix in 4% PFA for 5 minutes
5. Wash
6. Centrifuge at 1500 x g
7. Resuspend in PBS with 10% DMSO
8. Put in –80 freezer
9. Thaw
10. Stain (We do all of our "surface" markers during the permeabilization step)

Yes, you get very nice intracellular cytokines after one year.
See   http://www.ncbi.nlm.nih.gov/pubmed/21849680
All of the samples were processed in this manner.

I will email you a protocol off line.

Calman

From: Divya Bhatia <divyabhatia2 at gmail.com<mailto:divyabhatia2 at gmail.com>>
Date: Wednesday, April 3, 2013 8:11 AM
To: Calman Prussin <cprussin at niaid.nih.gov<mailto:cprussin at niaid.nih.gov>>
Cc: Sundararajan Jayaraman <anue2468 at uic.edu<mailto:anue2468 at uic.edu>>, Cytometry <cytometry at lists.purdue.edu<mailto:cytometry at lists.purdue.edu>>, "Lukas, Susan" <slukas at health.usf.edu<mailto:slukas at health.usf.edu>>
Subject: Re: [Cytometry] Frozen PBMCs for TBNK

Yes Prussin I usually cryopreserve alive cells, I would be thankful if you explain this protocol a bit. After isolation of pbmcs, you incubate the cells in 4 % paraformaldehyde for how long? how much in microliter should one incubate the cells with?then perform double wash with PBS,1X, i guess. Do you get signal of intracellular markers of Th1,Th2 and Th17 after thawing the cells stored for one year or so at -80 degree? The expression of CD62L and markers of neutrophils get downregulated after cryopreservation. What are the other markers, the expression of which get downregulated during the process of cryopreservation?
Thanks


On Wed, Apr 3, 2013 at 8:11 AM, Prussin, Calman (NIH/NIAID) [E] <CPRUSSIN at niaid.nih.gov<mailto:CPRUSSIN at niaid.nih.gov>> wrote:
[Boxbe]<https://www.boxbe.com/overview>[X] Prussin, Calman (NIH/NIAID) [E] (CPRUSSIN at niaid.nih.gov<mailto:CPRUSSIN at niaid.nih.gov>) is not on your Guest List<https://www.boxbe.com/approved-list?tc_serial=13881255596&tc_rand=889382870&utm_source=stf&utm_medium=email&utm_campaign=ANNO_MWTP&utm_content=001&token=shgyEPEBdxk8X9FiCEq0hLdZ765lTJJ1Vg1SvhPuATO3w%2BWVs%2BRanCocJ%2Bh1E2Zj&key=vvY9XtBqCx8XC57%2BuWBJNtBDVFTraNV8ODyHRqqhXqY%3D> | Approve sender<https://www.boxbe.com/anno?tc_serial=13881255596&tc_rand=889382870&utm_source=stf&utm_medium=email&utm_campaign=ANNO_MWTP&utm_content=001&token=shgyEPEBdxk8X9FiCEq0hLdZ765lTJJ1Vg1SvhPuATO3w%2BWVs%2BRanCocJ%2Bh1E2Zj&key=vvY9XtBqCx8XC57%2BuWBJNtBDVFTraNV8ODyHRqqhXqY%3D> | Approve domain<https://www.boxbe.com/anno?tc_serial=13881255596&tc_rand=889382870&utm_source=stf&utm_medium=email&utm_campaign=ANNO_MWTP&utm_content=001&dom&token=shgyEPEBdxk8X9FiCEq0hLdZ765lTJJ1Vg1SvhPuATO3w%2BWVs%2BRanCocJ%2Bh1E2Zj&key=vvY9XtBqCx8XC57%2BuWBJNtBDVFTraNV8ODyHRqqhXqY%3D>

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<http://tinyurl.com/prussin1>
calman at nih.gov<mailto:calman at nih.gov>
________________________________________
From: Divya Bhatia [divyabhatia2 at gmail.com<mailto:divyabhatia2 at gmail.com>]
Sent: Monday, April 01, 2013 1:10 AM
To: Sundararajan Jayaraman
Cc: cytometry at lists.purdue.edu<mailto: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<mailto: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><mailto:slukas at health.usf.edu<mailto:slukas at health.usf.edu>>
> >
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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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--
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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