[Cytometry] Frozen PBMCs for TBNK

Divya Bhatia divyabhatia2 at gmail.com
Wed Apr 3 08:11:31 EDT 2013


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> wrote:

>  [image: Boxbe] <https://www.boxbe.com/overview> Prussin, Calman
> (NIH/NIAID) [E] (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
> 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>
> > >
> > > -------------- next part --------------
> > > HTML attachment scrubbed and removed
> > > _______________________________________________
> > > Cytometry mailing list
> > > Cytometry at lists.purdue.edu
> > > https://lists.purdue.edu/mailman/listinfo/cytometry
> > > Search the list archive at  http://tinyurl.com/cytometry
> > >
> > >
> >
> >
> > _______________________________________________
> > Cytometry mailing list
> > Cytometry at lists.purdue.edu
> > https://lists.purdue.edu/mailman/listinfo/cytometry
> > Search the list archive at  http://tinyurl.com/cytometry
> >
>
>
>
> --
> *Thanks & Regards :
> Divya Bhatia
> Ph.D Student*
> *Department of Pediatrics
> All India Institute of Medical Sciences
> New Delhi, India*
> *Tel. 011-26546518*
> -------------- next part --------------
> HTML attachment scrubbed and removed
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
> Search the list archive at  http://tinyurl.com/cytometry
>
>


-- 
*Thanks & Regards :
Divya Bhatia
Ph.D Student*
*Department of Pediatrics
All India Institute of Medical Sciences
New Delhi, India*
*Tel. 011-26546518*
-------------- next part --------------
HTML attachment scrubbed and removed


More information about the Cytometry mailing list