Journal
NATURE BIOTECHNOLOGY
Volume 20, Issue 2, Pages 155-162Publisher
NATURE AMERICA INC
DOI: 10.1038/nbt0202-155
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Funding
- NIAID NIH HHS [AI35504, P01-AI39646] Funding Source: Medline
- NIAMS NIH HHS [AR44565, N01-AR-6-2227] Funding Source: Medline
- NIH HHS [A1/GF41520-01] Funding Source: Medline
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Intracellular assays of signaling systems have been limited by an inability to correlate functional subsets of cells in complex populations on the basis of active kinase states. Such correlations could be important in distinguishing changes in signaling status that arise in rare cell subsets during functional activation or in disease manifestation. Here we demonstrate the ability to simultaneously detect activated kinase members of the mitogen-activated protein kinases family (p38 MAPK, p44/42 MAPK, JNK/SAPK), members of cell survival pathways (AKT/PKB), and members of T-cell activation pathways (TYK2), among others, in subpopulations of complex cell populations by multiparameter flow-cytometric analysis. We demonstrate the utility of these probes in identifying distinct signaling cascades for (1) both artificial and physiological stimulatory conditions of peripheral blood mononuclear cells (PBMCs), (2) cytokine stimulation in human memory and naive lymphocyte subsets as identified by five differentiation markers, and (3) ordering of kinase activation in potential signaling hierarchies. Polychromatic flow-cytometric active kinase measurements demonstrate that multidimensional analysis of signaling pathways can provide functional signaling pathway assessment on a single-cell level and allow for potential correlation with biological and clinical parameters.
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