Re: TRAP-6 and plt activation

Gregor Rothe (gregor.rothe@klinik.uni-regensburg.de)
Tue, 18 Feb 1997 08:44:27 +0100

> On Mon, 17 Feb 1997 15:17:49 +0100, Jan Brozek asked:
>
> Can anybody help me in the matter of using TRAP-6 to activate platelets?
> How much TRAP do you use to maximally activate platelets? I mean how many
> micro mol per how many platelets? The papers I have read mention only the
> ammount of TRAP (usually 100uM).
> I had some problems with activating plts with TRAP - seems like I used too
> many plts, or too little TRAP, but maybe the problem is to be sought
> elswhere...

The increase in P-selectin will saturate with 20 uM/l TRAP-6
and after 10 min using 1:10 PBS diluted whole blood at RT.
If that does not work, what about organic solvents or azide
in your assay?

>
> The second question to the illuminati is how to determine the proper
> compensation for the WB 2 color platelet activation analysis?
>

Set a live-gate in forward versus side scatter on platelets and
compensate the fluorescence of either FITC or PE stained platelets to
that of unstained controls. Spectrally separated fluorochromes
e.g. PE and PerCP or FITC and PE/Cy5 are preferable due
to the high difference of staining intensity between activation
dependent (e.g. CD62P) and constitutive antigens (e.g. CD41)
which are useful for gating.

Gregor Rothe
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Institute for Clinical Chemistry and Laboratory Medicine
University of Regensburg
D-93042 Regensburg, Germany

Tel. +49 (941) 944-6204
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Internet: Gregor.Rothe@klinik.uni-regensburg.de
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