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Cytometry B Clin Cytom. 2017 May;92(3):207-210. doi: 10.1002/cyto.b.21207. Epub 2015 Apr 29.

Flow cytometric analysis of intracellular phosphoproteins in human monocytes.

Coppin E1,2,3,4, Malergue F5, Thibult ML1,2,3,4, Scifo C5, Favre C5, Nunès JA1,2,3,4.

Author information

1
Inserm, U1068, Centre de Recherche en Cancérologie de Marseille, Marseille, France.
2
Institut Paoli-Calmettes, Marseille, France.
3
CNRS, UMR7258, Centre de Recherche en Cancérologie de Marseille, Marseille, France.
4
Aix-Marseille Université, UM105, Marseille, France.
5
Beckman Coulter Immunotech, Life Sciences Global Assay and Applications Development, Marseille, France.

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.

KEYWORDS:

cell signaling; flow cytometry; monocytes; phosphoflow analysis; single cell analysis

PMID:
25914252
DOI:
10.1002/cyto.b.21207
[Indexed for MEDLINE]
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