Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry

Matthew R. Clutter, Garrett C. Heffner, Peter O. Krutzik, Kacey L. Sachen, Garry P. Nolan*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    21 Scopus citations

    Abstract

    Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios. © 2010 International Society for Advancement of Cytometry

    Original languageEnglish (US)
    Pages (from-to)1020-1031
    Number of pages12
    JournalCytometry Part A
    Volume77
    Issue number11
    DOIs
    StatePublished - Nov 2010

    Keywords

    • Background
    • Catalyzed reporter deposition
    • Cell signaling
    • Cell-based assay
    • Enzymatic amplification staining
    • Flow cytometry
    • Intracellular flow cytometry
    • Phospho flow
    • Signal transduction
    • Tyramide signal amplification

    ASJC Scopus subject areas

    • Pathology and Forensic Medicine
    • Histology
    • Cell Biology

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