Analysis of signaling events by dynamic phosphoflow cytometry

1Institut National de la Santé et de la Recherche Médicale, Unité 891, Centre de Recherche en Cancérologie de Marseille, F-13009 Marseille, France.
Science Signaling (Impact Factor: 7.65). 02/2009; 2(86):pl3. DOI: 10.1126/scisignal.286pl3
Source: PubMed

ABSTRACT Many proteins involved in cell signaling are phosphorylated. To determine the phosphorylation status of these signaling molecules at the single-cell level, we present a protocol for using state-specific antibodies to detect target phosphoproteins with fluorescence measurements by flow cytometry. To improve the signal intensity, a sandwich-labeling method for the analysis of signaling proteins is performed. By comparing the phosphorylation state of proteins in the presence and absence of sodium pervanadate, a nonspecific tyrosine phosphatase inhibitor, we determined the relative amount of tyrosine-phosphorylated protein in the samples, which reflects the activity of the signaling pathway. This dynamic approach, in combination with the signal amplification through a sandwich-labeling method, produces accurate and reproducible measurement of the activity of signaling pathways.

Download full-text


Available from: Jacques A Nunès, Jul 19, 2015
1 Follower
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Flow cytometry has revolutionized our ability to monitor immune responses by allowing us to simultaneously track a variety of cell surface and intracellular markers in discrete cell subsets in a highly sensitive and reproducible manner. This is especially critical in this new era of vaccinology trying to tackle the growing problems of chronic viral infections and cancer that not only evade host immune responses, but can negatively manipulate vaccine-induced immune responses. Thus, understanding how lymphocyte signaling is altered under normal and pathological conditions has become more critical. Over the last decade, a new flow cytometry technology called 'phosphoflow' (also sometimes called 'phosflow'), is rapidly developing for tracking multiple intracellular signaling molecules in the immune system at a single-cell level. Antibodies and reagents for tracking both tyrosine-phosphorylated and serine/threonine-phosphorylated signaling intermediaries in key immune signaling pathways have been developed, and phosphoflow is now starting to be applied to a wide variety of both preclinical and clinical studies on lymphocyte responses, as well as the functioning of cancer cells and virally infected cells. Here, we review the development of phosphoflow technology, its modern applications in the field of immunomonitoring and its current limitations. We then provide a perspective on the future of phosphoflow and a vision of how it can be applied to emerging critical questions in human vaccinology and public health.
    Expert Review of Vaccines 06/2010; 9(6):631-43. DOI:10.1586/erv.10.59 · 4.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphatidylinositol 5-phosphate (PtdIns5P) is emerging as a potential lipid messenger involved in several cell types, from plants to mammals. Expression of IpgD, a PtdIns(4,5)P(2) 4-phosphatase induces Src kinase and Akt, but not ERK activation and enhances interleukin II promoter activity in T-cells. Expression of a new PtdIns5P interacting domain blocks IpgD-induced T-cell activation and selective signaling molecules downstream of TCR triggering. Altogether, these data suggest that PtdIns5P may play a sensor function in setting the threshold of T-cell activation and contributing to maintain T-cell homeostasis.
    FEBS letters 06/2010; 584(11):2455-60. DOI:10.1016/j.febslet.2010.04.051 · 3.34 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Myeloproliferative neoplasms (MPNs) are associated with recurrent activating mutations of signaling proteins such as Janus kinase 2 (JAK2). However, the actual downstream signaling events and how these alter myeloid homeostasis are poorly understood. We developed an assay to measure basal levels of phosphorylated signaling intermediates by flow cytometry during myeloid differentiation in MPN patients. Our study provides the first systematic demonstration of specific signaling events and their comparison with disease phenotype and JAK2 mutation status. We demonstrate increased basal signaling in MPN patients, which occurs in both early and later stages of myeloid differentiation. In addition, the pattern of signaling is not correlated with JAK2 mutation status and signaling intensity is poorly correlated with mutant JAK2 allele burden. In contrast, signaling differences are detected between different MPN disease phenotypes. Finally, we demonstrate that signaling can be inhibited by a JAK2-selective small molecule, but that this inhibition is not JAK2 V617F specific, because MPN patients with mutant JAK2, wild-type JAK2, and control patients were inhibited to a similar degree. Our data suggest that, in addition to JAK2 mutations, other factors contribute significantly to the MPN phenotype, results that are relevant to both the pathogenesis and therapy of MPN.
    Blood 06/2011; 118(6):1610-21. DOI:10.1182/blood-2011-02-335042 · 10.43 Impact Factor
Show more