I have read about secreted IL-2 measurement as well as Erk, ZAP70 phosphorilation.... Ideally, I would like to measure only the activation generated through CD3Z (CD247) molecule signaling (which is integrated in TCR complex).
What about measureing CD25 expression by flow cytometry? This is general activation. For part two of the question: is there any ligand for CD3Z specifially? If it is part of the TCR complex, it will be hard to find a CD3Z specific read-out, since you are probably activating the whole TCR complex. Or is there any specific phosphorylation site on the intracellular side of CD3Z? Then you could do phosflow with a specific antibody for that site. There is one from BD: anti- pY142 (located at the intracellular domain of CD247) might do.
Alfonson,
To activate the T cell receptor, we use anti-CD3 antibodies (OKT3) either soluble or plate-bound. We usually measure TCR-induced activation by quantifying T cell output (IL-2, IFN-Y). You can also look at intracellular molecules activated by the TCR including phospho-zeta chain, Lck, Zap70, and LAT. As mentioned above, surface receptors will also give you a function of TCR-induced activation (i.e. CD69, CD44).
mesure thymidine incorporation after T-cell activation through anti-CD3 and anti-CD28.
Laurissa thymidine incorporation is used in the measurement of cell proliferation not activation.
thats very tricky ..since the CD247 (CD3zeta) Phosphorylation occurs anywhere between seconds to a minute..
I used an anti-phosphorylated CD247 antibody to measure the degree of activation
The cells have to be fixed immediately after stimulation in PFA 2% and then followed by harsh alcohol treatment (I used Phosflow III from BD) and then stained for pCD247 and surface markers (CD3, CD4 & CD8).
this can be used as starting guide
http://www.bdbiosciences.com/documents/Phosflow_Protocol_for_Human_Whole_Blood_Samples.pdf
Andrew, you are right but It is also known that activated T-cells proliferate. Nevertheless, the analysis of the expression of activation T-cells markers through QPCR, Cytometrie and sometimes WB are better to asses T-cell proliferation
The classical way is to measure IL-2 in the supernatant 18-24h post-stimulation by ELISA (the BD set works well), otherwise, a faster way is to stain for HLA-DR and CD25 and monitor the expression by FACS (don’t forget to stain unstimulated cells to help for gating).
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