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Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry

Nature Biotechnology volume 20, pages 155–162 (2002)Cite this article

Abstract

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 naïve 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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Figure 1: Phosphospecific antibodies correlate with kinase activity.
Figure 2: Multidimensional analysis provides insights into signaling networks as induced by differing stimuli.
Figure 3: Complexity of heterogenous kinase activation states is averaged upon cell sorting.
Figure 4: Flow diagram of resting T-cell isolation, preparation, and analysis by polychromatic flow cytometry (11-color, 13 parameter).
Figure 5: Multidimensional FACS analysis.
Figure 6: Signaling profiles are readily discernible in particular lymphocyte subsets using polychromatic flow cytometry.

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Acknowledgements

We are indebted to Steve De Rosa, Leonore A. Herzenberg, and Leonard A. Herzenberg for training in polychromatic flow cytometry. We also thank Gina Jager for flow cytometry preparation, Kevin Marks and Richard Smith for helpful and insightful discussions, and Mark Gilbert for FACStarPlus operation. This work was accomplished under a Scholar's award from the Leukemia and Lymphoma Society of America and a Burroughs Wellcome New Investigator in Pharmacology Award to G.P.N. The work was supported by NIH grants P01-AI39646, AR44565, AI35304, N01-AR-6-2227, A1/GF41520-01, and by the Juvenile Diabetes Foundation.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, 94305-5175, CA

    Omar D. Perez & Garry P. Nolan

  2. The Baxter Laboratory of Genetic Pharmacology, Stanford University School of Medicine, Stanford, 94305-5175, CA

    Omar D. Perez & Garry P. Nolan

  3. Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, 94305-5175, CA

    Omar D. Perez & Garry P. Nolan

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Correspondence to Garry P. Nolan.

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Perez, O., Nolan, G. Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol 20, 155–162 (2002). https://doi.org/10.1038/nbt0202-155

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