Flow cytometric analysis of intracellular phosphoproteins in human monocytes

Coppin, Emilie; Malergue, Fabrice; Thibult, Marie-Laure; Scifo, Caroline; Favre, C.; Nunès, Jacques A.

doi:10.1002/cyto.b.21207

Cited by 7 publications

(6 citation statements)

References 11 publications

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“…Analysis of alternations in cellular signaling induced by any one drug, combination of drugs, or the disease state can rapidly elucidate the intricate changes in cellular signaling which is crucial for determining how to therapeutically apply drugs for a proper balance between therapeutic outcomes and potential side effects. Phosphoflow technology is a robust tool that can monitor many cellular signaling pathways that can be modulated during drug treatment (George et al, 2015;Hildebrand and Kubatzky, 2017;Coppin et al, 2017). Using this technology one can rapidly interrogates the state of key signaling nodes in diverse cellular pathways.…”

Section: Discussionmentioning

confidence: 99%

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation

Krutzik

Ghaffari

et al. 2019

Antiviral Research

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A B S T R A C TAs antiviral drug resistance develops and new viruses emerge there is a pressing need to develop strategies to rapidly develop antiviral therapeutics. Here we use phospho-specific flow cytometry to assess perturbations of many different cellular signaling pathways during treatment with drug combinations that are highly effective in blocking Herpes simplex virus type 1 (HSV-1) infection. We discovered two antiviral drug combinations act on distinct signaling pathways, either STAT1 or S6 phosphorylation, to block HSV-1 infection. We focused on upregulation of S6 phosphorylation by HSV-1 infection, and our subsequent finding that ribavirin antagonizes this upregulation of S6 phosphorylation. We go on to show that the S6 kinase inhibitor SL0101 blocks HSV-1 replication in vitro and in an in vivo animal model of HSV-1 infection. Overall, we have used an unbiased analysis of cellular signaling pathways during treatment by antiviral drug combinations to discover a novel antiviral drug target against HSV-1 infection. The outcomes of the approach we present highlight the importance of analyzing how antiviral drugs modulate cellular and pathogen-induced signaling as a method to discover new drug therapy targets.

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Section: Discussionmentioning

confidence: 99%

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation

Krutzik

Ghaffari

et al. 2019

Antiviral Research

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“…In order to circumvent these limitations, it has been proposed to study cell signaling by flow cytometry [10]. This new technique, called phosflow, has rapidly gained interest over the last decade, based on the promise that it should allow tracking simultaneously multiple intracellular signaling molecules in the immune system at a single-cell level without the need for cell sorting or purification [11][12][13]. However, despite its many advantages, phosflow also have limitations.…”

Section: Introductionmentioning

confidence: 99%

Improved cell signaling analysis by biofunctionalized nanospheres and imaging flow cytometry

et al. 2021

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The analysis of immune cell signaling is critical for the understanding of the biology and pathology of the immune system, and thus a mandatory step for the development of efficient biomarkers and targeted therapies. Phosflow, which has progressively replaced the traditional western blot approach, relies on flow cytometry to analyze various signaling pathways at a single-cell level. This technique however suffers a lack of sensitivity largely due to the low signal/noise ratio that characterizes cell signaling analysis. In this study, we describe a new technique, which combines the use of biofunctionalized nanospheres (i.e., synthetic particulate antigens, SPAg) to stimulate the immune cells in suspension and imaging flow cytometry to identify homogenously-stimulated cells and quantify the activity of the chosen signaling pathway in selected subcellular regions of interest. Using BCR signaling as model, we demonstrate that SIBERIAN (SPAg-assIsted suB-cEllulaR sIgnaling ANalysis) allows assessing immune cell signaling with unprecedented sensitivity and specificity.

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“…Nevertheless, many other signaling pathways are involved in platelet pathophysiology and cannot be investigated with this kit. A new kit – PerFix EXPOSE – was recently released to measure by flow cytometry the degree of phosphorylation of various proteins in large cells as leukocytes and cell lines [2] , [3] . It was developed to enhance the signal-to-noise ratio of most intra-cellular phospho-epitopes stainings and to simplify the steps necessary for the sample preparation.…”

Section: Introductionmentioning

confidence: 99%

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples

Mallouk

Varvat

Berger

et al. 2018

Practical Laboratory Medicine

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A recently released kit (PerFix EXPOSE) was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP) phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12). The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24 h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP) of PGE1-induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets.

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Flow cytometric analysis of intracellular phosphoproteins in human monocytes

Abstract: This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society.

Cited by 7 publications

References 11 publications

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation

Cellular Signaling Analysis shows antiviral, ribavirin-mediated ribosomal signaling modulation

Improved cell signaling analysis by biofunctionalized nanospheres and imaging flow cytometry

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples

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