[Cytometry] Th-17 lymphocytes

Divya Bhatia divyabhatia2 at gmail.com
Thu Feb 6 01:29:42 EST 2014


Dear Elena,
I have been staining for Th17 population (CD3+CD8-IL-17+RORgT+) on
stimulated PBMCs (PMA=50 ng/ml; Ionomycin=500 ng/ml; Monensin=2 micromole)
and getting pretty good population.
The few events you are getting because you must be staining unstimulated
PBMCs.
If you want to use CD4,then pre-stain your PBMCs before stimulation with
PMA with anti-CD4
If you are okay with CD3+CD8- as Thelper population (although Th22 and
gamma delta are present,but the major % of cells are Th),you can perform
staining after stimulation also.
Make stock solutions of PMA,Ionomycin and Monensin/Brefeldin-A.
Use 10% RPMI-1640 complete media (90% RPMI, 10% FBS) for culture and
stimulation.
As you have been using kit so stimulate PBMCs and after that follow the
instructions for staining for Th17 as recommended in the kit protocol.
I have not used kit,but I think Monensin/Brefeldin-A (Golgi stop/Golgi
plug) must be provided along with the kit.
If even after that you do not get any event then try to standardize with
varying concentration as recommended in the spec sheet of the kit.

All the best


On Wed, Feb 5, 2014 at 12:31 AM, Elena Belyakova
<elena_belyakova at yahoo.com>wrote:

> Dear flow community,
>
> I need your help with measuring Th-17 lymphocytes in PBMC. One
> cardiologist from our medical centre start to perform a PhD about
> laboratory diagnostic of autoimmune myocarditis. She bought commercial kit
> for flow measuring Th-17 lymphocytes (
> http://www.biolegend.com/human-th17-flow-kit-cd3-fitc-cd4-pe-il-17-alexa-fluor-647-antibody-5654.html
> )
> Does anyone have an experience with this method? Because I've tried to do
> it several times, but there was just 1-3 events of h17+/CD3+/CD4+ on the
> dot plots! Cytokine staining protocol was the same as described at the web
> page for this product. And now  I'm a bit stuck as to how to perform this
> issue. Also I couldn't find reference range for Th-17 in blood.
> I was wondering if you could help me out, please. Sorry for my English.
>
> Kind regards,
> Elena Belyakova
> Federal Medical Investigation Centre named after Almazov
> St.Petersburg, Russia
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>


-- 


*Thanks & Regards :Divya BhatiaPh.D Student*


*Department of PediatricsAll India Institute of Medical SciencesNew Delhi,
India*
*Tel. 011-26546518*


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