PMID: 7545200Sep 11, 1995Paper

Flow cytometric analysis of protein-tyrosine phosphorylation in peripheral T cell subsets. Application to healthy and HIV-seropositive subjects

Journal of Immunological Methods
F VuillierG Dighiero

Abstract

This paper describes an improved method for detecting tyrosine phosphorylation levels in T cell subsets by flow cytometry early after CD3 crosslinking stimulation. It consists in introducing gentle paraformaldehyde fixation between CD3 crosslinking in cold conditions and the shift to 37 degrees C, which activates downstream signalling machinery. We used the combined properties of monoclonal antibodies for stimulating cells and for detecting surface markers to analyze protein-tyrosine phosphorylation levels in T cells subsets following stimulations which mimic physiological activation. Overall data obtained in healthy subjects, using two- or three-color immunofluorescence, indicated that: (1) most CD3 positive cells phosphorylate tyrosine substrates following CD3 crosslinking stimulation and (2) helper cells phosphorylate tyrosine to a slightly better extent than cytotoxic cells after CD3 crosslinking. Nevertheless, the two subsets follow similar kinetic patterns and tend to retain a homogeneous profile. Processing of samples from HIV-seropositive patients showed heterogeneous phosphorylation levels in both subsets, when compared to normal donors. This assay should, in the future, lead to easy and rapid exploration of the signal...Continue Reading

References

Nov 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·J J O'SheaM J Smyth
Oct 1, 1992·The Journal of Experimental Medicine·N K BandaT H Finkel
Apr 1, 1994·European Journal of Immunology·C R Maroun, M Julius

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Citations

Jun 14, 2005·Current Opinion in Immunology·Maria A SuniHolden T Maecker
Mar 13, 1998·Scandinavian Journal of Immunology·C MüllerO M Koch
Mar 4, 2010·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Marcelo HillEduardo Osinaga
Jun 4, 2010·Expert Review of Vaccines·Liubov ZaritskayaAnatoli M Malyguine
Jul 31, 1998·Leukemia & Lymphoma·C MüllerO M Koch

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