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

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.

SUBMITTER: Clutter MR

PROVIDER: S-EPMC3036012 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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

Clutter Matthew R MR Heffner Garrett C GC Krutzik Peter O PO Sachen Kacey L KL Nolan Garry P GP

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20101101 11

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 kina ...[more]

PMID: 20824632

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Project description:Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.

Dataset Information

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

Publications

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

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