[Cytometry] Th-17 lymphocytes

Karasyov, Artur Artur.Karasyov at cshs.org
Wed Feb 5 18:31:39 EST 2014


Hi Elena,

The sample dot plots on biolegend website are from stimulated PBMCs. Did
you stimulate your PBMCs before staining? We use final concentrations of
50ng/ml PMA and 1ug/ml ionomycin to stimulate PBMCs for 4 hours in a CO2
incubator. During the last 2 hours of stimulation we add berfeldin A to
trap IL-17a in the cells to detect intracellularly by flow. Together with
a similar staining protocol biolegend is recommending to use with their
kit, we can see a good number of IL-17a+ CD4 T cells most of the time. If
you did stimulate your sample and still don¹t see a good number of events,
it is not uncommon for PMA and Ionomycin to go bad.  Make sure those
reagents are good/fresh. To keep them ³good" use small aliquotes to avoid
too many freeze and thaw cycles on those reagents. Also, make sure you are
not exposing ionomycin to light for too long since it is light sensitive.
And Also, we keep the stock PMA and ionomycin resuspended in DMSO which is
the recommended solvent for both reagents I believe. Finally, make sure
you are acquiring many lymphocyte events when investigating Th17 since
they are a pretty rare population.

If you are talking about Th17 cell detection in PBMC without stimulation,
then, we also see very low number/percentage of CD3+/CD4+/IL-17a+ events
(roughly about 0.05-0.1% of CD3+ T cells in our tested samples) and I
don¹t know the reference range for Th-17 and would like to know as well.

Best of luck,

Artur Karasyov
Cedars-Sinai Medical Center
Los Angeles, CA

On 2/4/14, 11:01 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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