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Flow cytometric analysis of intracellular phosphoproteins in human monocytes.

Author information

Affiliations

  • 1. Inserm, U1068, Centre de Recherche en Cancérologie de Marseille, Marseille, France.
      Authors
    • Coppin E 1
    • Thibult ML 1
    • Nunès JA 1
    (3 authors)
  • 2. Beckman Coulter Immunotech, Life Sciences Global Assay and Applications Development, Marseille, France.
      Authors
    • Malergue F 2
    • Scifo C 2
    • Favre C 2
    (3 authors)

ORCIDs linked to this article

Cytometry. Part B, Clinical Cytometry, 29 Apr 2015, 92(3):207-210
https://doi.org/10.1002/cyto.b.21207 PMID: 25914252 

Abstract 

Background

Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation.

Methods

Whole blood was treated by lipopolysaccharide (LPS) or granulocyte-macrophage colony-stimulating factor (GM-CSF), then cells were labeled by antibodies recognizing cell surface and cytosolic proteins. Human monocytes were identified by a CD14 - CD45 staining and three phosphorylated proteins such as AKT, ERK-1/2, and STAT5, were simultaneously detected by multicolor phosphoflow analysis.

Results

By this rapid method, we are able to detect directly from a blood sample several signaling events in human monocytes where LPS stimulation induces preferentially ERK-1/2 phosphorylation where as GM-CSF stimulation induces STAT5 phosphorylation.

Conclusions

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

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Read article at publisher's site: https://doi.org/10.1002/cyto.b.21207

Read article for free, from open access legal sources, via Unpaywall: https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/cyto.b.21207Unpaywall

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Funding 

Funders who supported this work.

Fondation ARC pour la Recherche sur le Cancer

    Institut National Du Cancer (1)